Novel protein zlmda33

ABSTRACT

Novel polypeptides, polynucleotides encoding them, materials and methods for making them, antibodies that specifically bind to them, and methods of using the polypeptides, polynucleotides, and antibodies are disclosed. The polypeptides comprise at least nine contiguous amino acid residues of SEQ ID NO: 2 or SEQ ID NO: 5, and may be prepared as polypeptide fusions comprising heterologous sequences, such as affinity tags. The polypeptides and polynucleotides encoding them may be used within a variety of therepeutic, diagnostic, and research applications, including in vitro diagnosis and in vivo imaging of cancers and other sites of abnormal cell proliferation.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. 119(e) ofprovisional application No. 60/247,538, filed Nov. 9, 2000.

BACKGROUND OF THE INVENTION

[0002] Expression of specific genes and production of the cognateencoded proteins within cells and tissues is dependent in part upon themetabolic state of the cell or tissue. Certain genes are expressed onlyat specific developmental stages, such as the fetal stage. A protein maybe produced in one tissue during fetal development, and in anothertissue during the adult stage. Other proteins may be produced duringfetal development but not in normal adult tissue.

[0003] Genes may be expressed in abnormal amounts or abnormal locationsin some pathological states, particularly in cancers. Gene expressionand protein production are therefore indicators of the metabolic stateof cells and tissues. For example, production of a particular protein ina certain tissue may be indicative of a cancerous or pre-cancerousstate. Cancer-specific proteins may also be released by cells intobodily fluids, where they can be detected by conventional diagnosticmethods.

[0004] Cancer-specific gene expression also provides targets fortherapeutic intervention. By reducing or blocking such gene expressionor by interfering in the biological activity of the encoded protein,tumor development may be slowed or reversed. Tumor-specific proteinsalso provide targets for the delivery of therapeutic agents, providingfor more specific treatment with reduced side effects.

DESCRIPTION OF THE INVENTION

[0005] Within one aspect of the invention there is provided an isolatedpolypeptide comprising at least nine contiguous amino acid residues ofSEQ ID NO: 2 or SEQ ID NO: 5. Within one embodiment, the isolatedpolypeptide consists of from 15 to 1500 amino acid residues. Withinanother embodiment, the at least nine contiguous amino acid residues ofSEQ ID NO: 2 or SEQ ID NO: 5 are operably linked via a peptide bond orpolypeptide linker to a second polypeptide selected from the groupconsisting of maltose binding protein, an immunoglobulin constantregion, a polyhistidine tag, and a peptide as shown in SEQ ID NO: 3.Within another embodiment, the isolated polypeptide comprises at least30 contiguous residues of SEQ ID NO: 2 or SEQ ID NO: 5. Exemplarypolypeptides of the invention include, without limitation, thosecomprising residues 2-8, 15-22, 38-45, 94-99, 110-115, 196-202, 215-220,235-241, 2-22, 110-133, 120-133, 121-132, 192-203, 214-226, or 214-242SEQ ID NO: 2.

[0006] Within a second aspect of the invention there is provided anexpression vector comprising the following operably linked elements: atranscription promoter, a 10 DNA segment encoding a protein comprisingresidues 1-248 of SEQ ID NO: 2 or SEQ ID NO: 5, and a transcriptionterminator. Within one embodiment, the DNA segment comprises nucleotides174-917 of SEQ ID NO: 1. Within another embodiment, the expressionvector further comprises a secretory signal sequence operably linked tothe DNA segment.

[0007] Within a third aspect of the invention there is provided acultured cell into which has been introduced an expression vector asdisclosed above, wherein the cell expresses the DNA segment. Within oneembodiment, the expression vector comprises a secretory signal sequenceoperably linked to the DNA segment, and the protein is secreted by thecell. The cultured cell of the invention can be used, inter alia, withina method of making a protein, wherein the cell is cultured underconditions whereby the DNA segment is expressed and the protein isproduced, and the protein is recovered. Within one embodiment, theexpression vector comprises a secretory signal sequence operably linkedto the DNA segment, the protein is secreted by the cell, and the proteinis recovered from a medium in which the cell is cultured.

[0008] Within a fourth aspect of the invention there is provided aprotein produced by the method disclosed above.

[0009] Within a fifth aspect of the invention there is provided anantibody that specifically binds to a protein as disclosed above. Withinone embodiment of the invention the antibody is labeled to produce adetectable signal.

[0010] Within a sixth aspect of the invention there is provided a methodof detecting, in a test sample, a polypeptide selected from the groupconsisting of (a) a polypeptide as shown in SEQ ID NO: 2, (b) apolypeptide as shown in SEQ ID NO: 5, and (c) proteolytic fragments of(a) or (b). The method comprises combining the test sample with anantibody as disclosed above under conditions whereby the antibody bindsto the polypeptide, and detecting the presence of antibody bound to thepolypeptide.

[0011] Within a seventh aspect of the invention there are providedisolated polynucleotides encoding the polypeptides disclosed above.Within one aspect of the invention the isolated polynucleotide comprisesnucleotides 1-744 of SEQ ID NO: 4 or SEQ ID NO: 6. Within another aspectof the invention the isolated polynucleotide comprises nucleotides174-917 of SEQ ID NO: 1.

[0012] These and other aspects of the invention will become evident uponreference to the following detailed description of the invention and theaccompanying FIGURE.

[0013] The FIGURE is a Kyte-Doolittle hydrophilicity profile of theamino acid sequence shown in SEQ ID NO: 2. The profile was preparedusing Protean™ 3.14 (DNAStar, Madison, Wis.).

[0014] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms:

[0015] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985) (SEQID NO: 3), substance P, Flag™ peptide (Hopp et al., Biotechnology6:1204-1210, 1988), streptavidin binding peptide, maltose bindingprotein (Guan et al., Gene 67:21-30, 1987), cellulose binding protein,thioredoxin, ubiquitin, T7 polymerase, or other antigenic epitope orbinding domain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags and otherreagents are available from commercial suppliers (e.g., PharmaciaBiotech, Piscataway, N.J.; New England Biolabs, Beverly, Mass.; EastmanKodak, New Haven, Conn.).

[0016] The term “allelic variant” is used herein to denote any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequences. The term allelic variant is also usedherein to denote a polypeptide encoded by an allelic variant of a gene.

[0017] A “complement” of a polynucleotide molecule is a polynucleotidemolecule having a complementary base sequence and reverse orientation ascompared to a reference sequence. For example, the sequence 5′ ATGCACGGG3′ is complementary to 5′ CCCGTGCAT 3′.

[0018] “Conservative amino acid substitutions” are defined by theBLOSUM62 scoring matrix of Henikoff and Henikoff, Proc. Natl. Acad. Sci.USA 89:10915-10919, 1992, an amino acid substitution matrix derived fromabout 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins. As used herein, the term “conservative amino acidsubstitution” refers to a substitution represented by a BLOSUM62 valueof greater than −1. For example, an amino acid substitution isconservative if the substitution is characterized by a BLOSUM62 value of0, 1, 2, or 3. Preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least one 1 (e.g., 1, 2 or 3),while more preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

[0019] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0020] The term “expression vector” is used to denote a DNA molecule,linear or circular, that comprises a segment encoding a polypeptide ofinterest operably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

[0021] An “inhibitory polynucleotide” is a DNA or RNA molecule thatreduces or prevents expression (transcription or translation) of asecond (target) polynucleotide. Inhibitory polynucleotides include antisense polynucleotides, ribozymes, and external guide sequences. The term“inhibitory polynucleotide” further includes DNA and RNA molecules thatencode the actual inhibitory species, such as DNA molecules that encoderibozymes.

[0022] The term “isolated”, when applied to a polynucleotide, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985).

[0023] An “isolated” polypeptide or protein is a polypeptide or proteinthat is found in a condition other than its native environment, such asapart from blood and animal tissue. The isolated polypeptide or proteinmay be prepared substantially free of other polypeptides or proteins,particularly those of animal origin. For some purposes, the polypeptidesand proteins will be prepared in a highly purified form, i.e. greaterthan 95% pure or greater than 99% pure. When used in this context, theterm “isolated” does not exclude the presence of the same polypeptide orprotein in alternative physical forms, such as dimers or alternativelyglycosylated or derivatized forms.

[0024] “Operably linked” means that two or more entities are joinedtogether such that they function in concert for their intended purposes.When referring to DNA segments, the phrase indicates, for example, thatcoding sequences are joined in the correct reading frame, andtranscription initiates in the promoter and proceeds through the codingsegment(s) to the terminator. When referring to polypeptides, “operablylinked” includes both covalently (e.g., by disulfide bonding) andnon-covalently (e.g., by hydrogen bonding, hydrophobic interactions, orsalt-bridge interactions) linked sequences, wherein the desiredfunction(s) of the sequences are retained.

[0025] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0026] A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theseterms are applied to double-stranded molecules they are used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in 5 length.

[0027] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0028] The term “promoter” is used herein for its art-recognized meaningto denote a portion of a gene containing DNA sequences that provide forthe binding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

[0029] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless. Thus, a protein “consisting of”, for example, from 15 to1500 amino acid residues may further contain one or more carbohydratechains.

[0030] A “secretory signal sequence” is a DNA sequence that encodes apolypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

[0031] A “segment” is a portion of a larger molecule (e.g.,polynucleotide or polypeptide) having specified attributes. For example,a DNA segment encoding a specified polypeptide is a portion of a longerDNA molecule, such as a plasmid or plasmid fragment, that, when readfrom the 5′ to the 3′ direction, encodes the sequence of amino acids ofthe specified polypeptide.

[0032] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

[0033] All references cited herein are incorporated by reference intheir entirety.

[0034] The present invention is based on the discovery of a novelpolynucleotide and protein encoded by the polynucleotide. Thepolynucleotide is expressed primarily in fetal brain, spinal cord,testis prostate smooth muscle cells, thyroid, bone marrow, uterinetumor, and ovarian tumor, and is not expressed in many other tissues andcells, including normal uterus and normal ovary. The polynucleotide andprotein are thus markers for uterine and ovarian tumors in a mammal, andalso provide targets for diagnostic and therapeutic agents. This novelprotein is termed “zlmda33.”

[0035] A representative human zlmda33 DNA sequence is shown in SEQ IDNO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2. Thehuman protein comprises 248 amino acid residues. It does not appear tocontain a secretory peptide, and is therefore believed to be anintracellular protein. Those skilled in the art will recognize that SEQID NO: 1 and SEQ ID NO: 2 represent a single allele of zlmda33, and thatallelic variation is expected to exist. A second human zlmda33 proteinis shown in SEQ ID NO: 5, which differs from SEQ ID NO: 2 at residues 85and 111. Those skilled in the art will also recognize that many proteinsare produced in alternatively spliced forms.

[0036] Polypeptides of the present invention comprise at least 9 or atleast 15 contiguous amino acid residues of SEQ ID NO: 2 or SEQ ID NO: 5.Within certain embodiments of the invention, the polypeptides comprise20, 30, 40, 50, 100, or more contiguous residues of SEQ ID NO: 2 or SEQID NO: 5, up to the entire primary translation product (residues 1 to248 of SEQ ID NO: 2 or SEQ ID NO: 5). As disclosed in more detail below,these polypeptides can further comprise additional, non-zlmda33,polypeptide sequence(s).

[0037] Within the polypeptides of the present invention are polypeptidesthat 3 0 comprise an epitope-bearing portion of a protein as shown inSEQ ID NO: 2 or SEQ ID NO: 5. An “epitope” is a region of a protein towhich an antibody can bind. See, for example, Geysen et al., Proc. Natl.Acad. Sci. USA 81:3998-4002, 1984. Epitopes can be linear orconformational, the latter being composed of discontinuous regions ofthe protein that form an epitope upon folding of the protein. Linearepitopes are generally at least 6 amino acid residues in length.Relatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, Sutcliffe et al., Science219:660-666, 1983. Antibodies that recognize short, linear epitopes areparticularly useful in analytic and diagnostic applications that employdenatured protein, such as Western blotting (Tobin, Proc. Natl. Acad.Sci. USA 76:4350-4356, 1979), or in the analysis of fixed cells ortissue samples. Antibodies to linear epitopes are also useful fordetecting fragments of zlmda33, such as might occur in body fluids orcell culture media.

[0038] Antigenic, epitope-bearing polypeptides of the present inventionare useful for raising antibodies, including monoclonal antibodies, thatspecifically bind to a zlmda33 protein. Antigenic, epitope-bearingpolypeptides contain a sequence of at least six, generally at leastnine, often from 15 to about 30 contiguous amino acid residues of azlmda33 protein (e.g., SEQ ID NO: 2). Polypeptides comprising a largerportion of a zlmda33 protein, i.e. from 30 to 50 residues up to theentire sequence, are included. It is preferred that the amino acidsequence of the epitope-bearing polypeptide is selected to providesubstantial solubility in aqueous solvents, that is the sequenceincludes relatively hydrophilic residues, and hydrophobic residues aresubstantially avoided. Such regions include those comprising residues2-8, 15-22, 38-45, 94-99, 110-115, 121-132, 196-202, 215-220, and235-241 of SEQ ID NO: 2. Larger hydrophilic peptides include, forexample, residues 2-22, 110-133, 120-133, 192-203, 214-226, and 214-242.

[0039] Polypeptides of the present invention can be prepared with one ormore amino acid substitutions, deletions or additions as compared to SEQID NO: 2 or SEQ ID NO: 5. These changes are preferably of a minornature, such as conservative amino acid substitutions and other changesthat do not significantly affect the folding or activity of the proteinor polypeptide. Other exemplary changes include amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, an amino or carboxyl-terminal cysteine residue to facilitatesubsequent linking to maleimide-activated keyhole limpet hemocyanin, asmall linker peptide of up to about 20-25 residues, or an extension thatfacilitates purification (an affinity tag) as disclosed above. Two ormore affinity tags may be used in combination. Polypeptides comprisingaffinity tags can further comprise a polypeptide linker and/or aproteolytic cleavage site between the zlmda33 polypeptide and theaffinity tag. Such cleavage sites include, for example, thrombincleavage sites and factor Xa cleavage sites.

[0040] The present invention further provides a variety of otherpolypeptide fusions. For example, a zlmda33 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Dimerizing proteins in this regard include, forexample, immunoglobulin fragments comprising constant region and hingedomains. For example, a zlmda33 polypeptide can be joined to an IgG Fcfragment (consisting essentially of C_(H)2, C_(H)3, and hinge). Suchfusions are typically secreted as multimeric molecules wherein the Fcportions are disulfide bonded to each other and the two non-Igpolypeptides are arrayed in close proximity to each other. Dimerizationcan also be stabilized by fusing a zlmda33 polypeptide to a leucinezipper sequence (Riley et al., Protein Eng. 9:223-230, 1996; Mohamed etal., J. Steroid Biochem. Mol. Biol. 51:241-250, 1994).Immunoglobulin-zlmda33 polypeptide fusions and leucine zipper fusionscan be expressed in genetically engineered cells to produce a variety ofmultimeric zlmda33 analogs. Auxiliary domains can be fused to zlmda33polypeptides to target them to specific cells, tissues, ormacromolecules (e.g., collagen). For example, a zlmda33 polypeptide orprotein can be targeted to a predetermined cell type by fusing a zlmda33polypeptide to a ligand that specifically binds to a receptor on thesurface of the target cell. In this way, polypeptides and proteins canbe targeted for therapeutic or diagnostic purposes. A zlmda33polypeptide can be fused to two or more moieties, such as an affinitytag for purification and a targeting domain. Polypeptide fusions canalso comprise one or more cleavage sites, particularly between domains.See, Tuan et al., Connective Tissue Research 34:1-9, 1996. Withinimmunoglobulin-zlmda33 fusion proteins, certain amino acidsubsititutions may be introduced into the Ig portion to alter effectorfunctions associated with the native Ig. For example, amino acidsubstitutions can be made at EU index positions 234, 235, and 237 toreduce binding to FcγRI, and at EU index positions 330 and 331 to reducecomplement fixation. See, Duncan et al., Nature 332:563-564, 1988;Winter et al., U.S. Pat. No. 5,624,821; Tao et al., J. Exp. Med.178:661, 1993; and Canfield and Morrison, J. Exp. Med. 173:1483, 1991.The carboxyl-terminal lysine residue can be removed from the C_(H)3domain to increase homogeneity of the product. Within fusions to an Igheavy chain polypeptide, the Cys residue within the hinge region that isordinarily disulfide-bonded to the light chain can be replaced withanother amino acid residue, such as a serine residue, if the Ig fusionis not co-expressed with a light chain polypeptide. However, anIg-zlmda33 fusion polypeptide can be co-expressed with a wild-type orfused light chain polypeptide as disclosed in U.S. Pat. No. 6,018,026.In addition, a zlmda33 polypeptide can be joined to another bioactivemolecule, such as a cytokine, to provide a multi-functional molecule.

[0041] Polypeptide fusions of the present invention will generallycontain not more than about 1,500 amino acid residues, usually not morethan about 1,300 residues, more commonly not more than about 1,000residues, and will in many cases be considerably smaller. For example, azlmda33 polypeptide of 248 residues (e.g., residues 1-248 of SEQ ID NO:2) can be fused to E. coli β-galactosidase (1,021 residues; seeCasadaban et al., J. Bacteriol. 143:971-980, 1980), a 10-residue spacer,and a 4-residue factor Xa cleavage site to yield a polypeptide of 1,283residues. In a second example, residues 1-248 of SEQ ID NO: 2 can befused to maltose binding protein (approximately 370 residues), a4-residue cleavage site, and a 6-residue polyhistidine tag.

[0042] The proteins of the present invention can also comprisenon-naturally occuring amino acid residues. Non-naturally occuring aminoacids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occuring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNAs are known in the art. Transcription and translationof plasmids containing nonsense mutations is carried out in a cell-freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science259:806-809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145-10149, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-19998, 1996). Within a third method, E. coli cells arecultured in the absence of a natural amino acid that is to be replaced(e.g., phenylalanine) and in the presence of the desired non-naturallyoccuring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccuring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occuring amino acid residues can be converted to non-naturallyoccuring species by in vitro chemical modification. Chemicalmodification can be combined with site-directed mutagenesis to furtherexpand the range of substitutions (Wynn and Richards, Protein Sci.2:395-403, 1993).

[0043] Amino acid sequence changes are made in zlmda33 polypeptides soas to minimize disruption of higher order structure essential tobiological activity. Amino acid residues that are within regions ordomains that are critical to maintaining structural integrity can bedetermined. Within these regions one can identify specific residues thatwill be more or less tolerant of change and maintain the overalltertiary structure of the molecule. Methods for analyzing sequencestructure include, but are not limited to, alignment of multiplesequences with high amino acid or nucleotide identity, secondarystructure propensities, binary patterns, complementary packing, andburied polar interactions (Barton, Current Opin. Struct. Biol.5:372-376, 1995 and Cordes et al., Current Opin. Struct. Biol. 6:3-10,1996). In general, determination of structure will be accompanied byevaluation of activity of modified molecules. The effects of amino acidsequence changes can be predicted by, for example, computer modelingusing available software (e.g., the Insight II® viewer and homologymodeling tools; MSI, San Diego, Calif.) or determined by analysis ofcrystal structure (see, e.g., Lapthorn et al, Nature 369:455-461, 1994;Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). Protein foldingcan be measured by circular dichroism (CD). Measuring and comparing theCD spectra generated by a modified molecule and standard molecule areroutine in the art (Johnson, Proteins 7:205-214, 1990). Crystallographyis another well-known and accepted method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are other known methods for analyzing folding andstructural similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992). Mass spectrometry and chemicalmodification using reduction and alkylation can be used to identifycysteine residues that are associated with disulfide bonds or are freeof such associations (Bean et al., Anal. Biochem. 201:216-226, 1992;Gray, Protein Sci. 2:1732-1748, 1993; and Patterson et al., Anal. Chem.66:3727-3732, 1994). Alterations in disulfide bonding will be expectedto affect protein folding. These techniques can be employed individuallyor in combination to analyze and compare the structural features thataffect folding of a variant protein or polypeptide to a standardmolecule to determine whether such modifications would be significant.

[0044] A hydrophilicity profile of SEQ ID NO: 2 is shown in the attachedfigure. Those skilled in the art will recognize that hydrophilicity willbe taken into account when designing alterations in the amino acidsequence of a zlmda33 polypeptide, so as not to disrupt the overallprofile.

[0045] Essential amino acids in the polypeptides of the presentinvention can be identified experimentally according to procedures knownin the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989; Bass etal., Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991). In the lattertechnique, single alanine mutations are introduced throughout themolecule, and the resultant mutant molecules are tested for biologicalactivity as disclosed below to identify amino acid residues that arecritical to the activity of the molecule.

[0046] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). These authors disclosemethods for simultaneously randomizing two or more positions in apolypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

[0047] Variants of the disclosed zlmda33 DNA and polypeptide sequencescan be generated through DNA shuffling as disclosed by Stemmer, Nature370:389-391, 1994 and Stemmer, Proc. Natl. Acad. Sci. USA91:10747-10751, 1994. Briefly, variant genes are generated by in vitrohomologous recombination by random fragmentation of a parent genefollowed by reassembly using PCR, resulting in randomly introduced pointmutations. This technique can be modified by using a family of parentgenes, such as allelic variants or genes from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

[0048] In many cases, the structure of the final polypeptide productwill result from processing of the nascent polypeptide chain by the hostcell, thus the final sequence of a zlmda33 polypeptide produced by ahost cell will not always correspond to the full sequence encoded by theexpressed polynucleotide. Such processing events include, for example,proteolysis, carbohydrate addition, and amino acid side chainmodification. Differential processing of individual chains may result inheterogeneity of expressed polypeptides.

[0049] Mutagenesis methods as disclosed above can be combined with highvolume or high-throughput screening methods to detect biologicalactivity of zlmda33 variant polypeptides. Assays that can be scaled upfor high throughput include mitogenesis assays, which can be run in a96-well format. Mutagenized DNA molecules that encode active zlmda33polypeptides can be recovered from the host cells and rapidly sequencedusing modern equipment. These methods allow the rapid determination ofthe importance of individual amino acid residues in a polypeptide ofinterest, and can be applied to polypeptides of unknown structure.

[0050] Using the methods discussed above, one of ordinary skill in theart can prepare a variety of polypeptide fragments or variants of SEQ IDNO: 2 or SEQ ID NO: 5 that retain the activity of wild-type zlmda33.

[0051] The present invention also provides zlmda33 polynucleotidemolecules. These polynucleotides include DNA and RNA, both single- anddouble-stranded, the former encompassing both the sense strand and theantisense strand. A representative DNA sequence encoding the amino acidsequence of SEQ ID NO: 2 is shown in SEQ ID NO: 1. Those skilled in theart will readily recognize that, in view of the degeneracy of thegenetic code, considerable sequence variation is possible among thesepolynucleotide molecules. SEQ ID NO: 4 is a degenerate DNA sequence thatencompasses all DNAs that encode the zlmda33 polypeptide of SEQ ID NO:2. SEQ ID NO: 6 is a degenerate DNA sequence that encompasses all DNAsthat encode the zlmda33 polypeptide of SEQ ID NO: 5. Those skilled inthe art will recognize that the degenerate sequences of SEQ ID NO: 4 andSEQ ID NO: 6 also provides all RNA sequences encoding SEQ ID NO: 2 andSEQ ID NO: 5, respectively, by substituting U for T. Thus, zlmda33polypeptide-encoding polynucleotides comprising nucleotides 1-744 of SEQID NO: 4, SEQ ID NO: 6, and their RNA equivalents are contemplated bythe present invention, as are segments of SEQ ID NO: 4 and SEQ ID NO: 6encoding other zlmda33 polypeptides disclosed herein. Table 1 sets forththe one-letter codes used within SEQ ID NO: 4 and SEQ ID NO: 6 to denotedegenerate nucleotide positions. “Resolutions” are the nucleotidesdenoted by a code letter. “Complement” indicates the code for thecomplementary nucleotide(s). For example, the code Y denotes either C orT, and its complement R denotes A or G, A being complementary to T, andG being complementary to C. TABLE 1 Nucleotide Resolutions ComplementResolutions A A T T C C G G G G C C T T A A R A|G Y C|T Y C|T R A|G MA|C K G|T K G|T M A|C S C|G S C|G W A|T W A|T H A|C|T D A|G|T B C|G|T VA|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T

[0052] The degenerate codons used in SEQ ID NO: 4 and SEQ ID NO: 6,encompassing all possible codons for a given amino acid, are set forthin Table 2, below. TABLE 2 One- Amino Letter Degenerate Acid Code CodonsCodon Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACCACG ACT CAN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly GGGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAGGAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGTMGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTCCTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr YTAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR Asn{Asp B RAY Glu{Gin ZSAR Any X NNN Gap — - - -

[0053] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by a degenerate sequencemay encode variant amino acid sequences, but one of ordinary skill inthe art can easily identify such variant sequences by reference to theamino acid sequence of SEQ ID NO: 2. Variant sequences can be readilytested for functionality as described herein.

[0054] One of ordinary skill in the art will also appreciate thatdifferent species can exhibit preferential codon usage. See, in general,Grantham et al., Nuc. Acids Res. 8:1893-1912, 1980; Haas et al. Curr.Biol. 6:315-324, 1996; Wain-Hobson et al., Gene 13:355-364, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-3087, 1986; and Ikemura, J. Mol. Biol. 158:573-597, 1982.Introduction of preferred codon sequences into recombinant DNA can, forexample, enhance production of the protein by making protein translationmore efficient within a particular cell type or species. Therefore, thedegenerate codon sequence disclosed in SEQ ID NO: 4 serves as a templatefor optimizing expression of polynucleotides in various cell types andspecies commonly used in the art and disclosed herein.

[0055] Within certain embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO: 1or a sequence complementary thereto under stringent conditions. Ingeneral, stringent conditions are selected to be about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Typical stringent conditions are those in whichthe salt concentration is up to about 0.03 M at pH 7 and the temperatureis at least about 60° C.

[0056] As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of zlmda33 RNA. Cells from testis, spinalcord, fetal brain, ovarian tumor, and uterine tumor are preferred. TotalRNA can be prepared using guanidine HCl extraction followed by isolationby centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNA using themethod of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-1412,1972). Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA usingknown methods. In the alternative, genomic DNA can be isolated.Polynucleotides encoding zlmda33 polypeptides are then identified andisolated by, for example, hybridization or PCR.

[0057] Full-length clones encoding zlmda33 can be obtained byconventional cloning procedures. Complementary DNA (cDNA) clones areusually preferred, although for some applications (e.g., expression intransgenic animals) it may be preferable to use a genomic clone, or tomodify a cDNA clone to include at least one genomic intron. Methods forpreparing cDNA and genomic clones are well known and within the level ofordinary skill in the art, and include the use of the sequence disclosedherein, or parts thereof, for probing or priming a library. Expressionlibraries can be probed with antibodies to zlmda33, receptor fragments,or other specific binding partners.

[0058] Zlmda33 polynucleotide sequences disclosed herein can also beused as probes or primers to clone 5′ non-coding regions of a zlmda33gene. Promoter elements from a zlmda33 gene can be used to direct theexpression of heterologous genes in, for example, transgenic animals orpatients treated with gene therapy. Cloning of 5′ flanking sequencesalso facilitates production of zlmda33 proteins by “gene activation” asdisclosed in U.S. Pat. No. 5,641,670. Briefly, expression of anendogenous zlmda33 gene in a cell is altered by introducing into thezlmda33 locus a DNA construct comprising at least a targeting sequence,a regulatory sequence, an exon, and an unpaired splice donor site. Thetargeting sequence is a zlmda33 5′ non-coding sequence that permitshomologous recombination of the construct with the endogenous zlmda33locus, whereby the sequences within the construct become operably linkedwith the endogenous zlmda33 coding sequence. In this way, an endogenouszlmda33 promoter can be replaced or supplemented with other regulatorysequences to provide enhanced, tissue-specific, or otherwise regulatedexpression. A portion of the zlmda33 gene, including a large 5′non-coding region, is available as GenBank accession number AF189745.

[0059] Those skilled in the art will recognize that the sequencesdisclosed in SEQ ID NOS:1 and 2 represent a single allele of humanzlmda33. Allelic variants of these sequences can be cloned by probingcDNA or genomic libraries from different individuals according tostandard procedures.

[0060] The present invention further provides counterpart polypeptidesand polynucleotides from other species (“orthologs”). Of particularinterest are zlmda33 polypeptides from other mammalian species,including murine, porcine, ovine, bovine, canine, feline, equine, andother primate polypeptides. These non-human zlmda33 polypeptides andpolynucleotides, as well as antagonists thereof and other relatedmolecules, can be used, inter alia, in veterinary medicine. Orthologs ofhuman zlmda33 can be cloned using information and compositions providedby the present invention in combination with conventional cloningtechniques. For example, a cDNA can be cloned using mRNA obtained from atissue or cell type that expresses zlmda33 as disclosed above. A libraryis then prepared from mRNA of a positive tissue or cell line. Azlmda33-encoding cDNA can then be isolated by a variety of methods, suchas by probing with a complete or partial human cDNA or with one or moresets of degenerate probes based on the disclosed sequence. A cDNA canalso be cloned using the polymerase chain reaction, or PCR (Mullis, U.S.Pat. No. 4,683,202), using primers designed from the representativehuman zlmda33 sequence disclosed herein. Within an additional method, acDNA library can be used to transform or transfect host cells, andexpression of the cDNA of interest can be detected with an antibody tozlmda33 polypeptide. Similar techniques can also be applied to theisolation of genomic clones.

[0061] For any zlmda33 polypeptide, including variants and fusionproteins, one of ordinary skill in the art can readily generate a fullydegenerate polynucleotide sequence encoding that variant using theinformation set forth in Tables 1 and 2, above. Moreover, those of skillin the art can use standard software to devise zlmda33 variants basedupon the nucleotide and amino acid sequences described herein. Thepresent invention thus provides a computer-readable medium encoded witha data structure that provides at least one of the following sequences:SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, and portionsthereof. Suitable forms of computer-readable media include, withoutlimitation, a hard or fixed drive, a random access memory (RAM) chip, afloppy disk, digital linear tape (DLT), a disk cache, a ZIP™ disk,compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW), andCD-recordable), digital versatile/video discs (DVD) (e.g., DVD-ROM,DVD-RAM, and DVD+RW), and carrier waves.

[0062] The zlmda33 polypeptides of the present invention, includingfull-length polypeptides, biologically active fragments, and fusionpolypeptides can be produced according to conventional techniques usingcells into which have been introduced an expression vector encoding thepolypeptide. As used herein, “cells into which have been introduced anexpression vector” include both cells that have been directlymanipulated by the introduction of exogenous DNA molecules and progenythereof that contain the introduced DNA. Suitable host cells are thosecell types that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Techniques for manipulating cloned DNAmolecules and introducing exogenous DNA into a variety of host cells aredisclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, and Ausubel et al., eds., Current Protocols in Molecular Biology,John Wiley and Sons, Inc., N.Y., 1987.

[0063] In general, a DNA sequence encoding a zlmda33 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers can be provided on separate vectors, and replicationof the exogenous DNA is provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

[0064] To direct a zlmda33 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be derived from another secretedprotein (e.g., t-PA; see, U.S. Pat. No. 5,641,655) or synthesized denovo. The secretory signal sequence is operably linked to the zlmda33DNA sequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly sythesized polypeptide into thesecretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain signal sequences may be positioned elsewherein the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

[0065] Cultured mammalian cells can be used as hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993). The production of recombinant polypeptides in cultured mammaliancells is disclosed by, for example, Levinson et al., U.S. Pat. No.4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S.Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitablecultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7(ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72,1977) and Chinese hamster ovary (e.g. CHO-K1, ATCC No. CCL 61; or CHODG44, Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986) cell lines.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Manassas, Va. Suitable promoters include those from metallothioneingenes (U.S. Pat. Nos. 4,579,821 and 4,601,978), the adenovirus majorlate promoter, and promoters from SV-40 or cytomegalovirus. See, e.g.,U.S. Pat. Nos. 4,579,821; 4,601,978; and 4,956,288. Expression vectorsfor use in mammalian cells include pZP-1 and pZP-9, which have beendeposited with the American Type Culture Collection, Manassas, Va. USAunder accession numbers 98669 and 98668, respectively, and derivativesthereof.

[0066] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Anexemplary selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.An exemplary amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that produce analtered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, and placental alkalinephosphatase, can be used to sort transfected cells from untransfectedcells by such means as FACS or magnetic bead separation technology.

[0067] The adenovirus system (disclosed in more detail below) can alsobe used for protein production in vitro. By culturingadenovirus-infected non-293 cells under conditions where the cells arenot rapidly dividing, the cells can produce proteins for extendedperiods of time. For instance, BHK cells are grown to confluence in cellfactories, then exposed to the adenoviral vector encoding the secretedprotein of interest. The cells are then grown under serum-freeconditions, which allows infected cells to survive for several weekswithout significant cell division. In an alternative method, adenovirusvector-infected 293 cells can be grown as adherent cells or insuspension culture at relatively high cell density to producesignificant amounts of protein (See Gamier et al., Cytotechnol.15:145-55, 1994). With either protocol, an expressed, secretedheterologous protein can be repeatedly isolated from the cell culturesupernatant, lysate, or membrane fractions depending on the dispositionof the expressed protein in the cell. Within the infected 293 cellproduction protocol, non-secreted proteins can also be effectivelyobtained.

[0068] Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa califomica nuclear polyhedrosis virus(AcNPV) according to methods known in the art, such as thetransposon-based system described by Luckow et al. (J. Virol.67:4566-4579, 1993). This system, which utilizes transfer vectors, iscommercially available in kit form (Bac-to-Ba™ kit; Life Technologies,Rockville, Md.). The transfer vector (e.g., pFastBac1™; LifeTechnologies) contains a Tn7 transposon to move the DNA encoding theprotein of interest into a baculovirus genome maintained in E. coli as alarge plasmid called a “bacmid.” See, Hill-Perkins and Possee, J. Gen.Virol. 71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556,1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549, 1995.In addition, transfer vectors can include an in-frame fusion with DNAencoding a polypeptide extension or affinity tag as disclosed above.Using techniques known in the art, a transfer vector containing azlmda33-encoding sequence is transformed into E. coli host cells, andthe cells are screened for bacmids which contain an interrupted lacZgene indicative of recombinant baculovirus. The bacmid DNA containingthe recombinant baculovirus genome is isolated, using common techniques,and used to transfect Spodoptera frugiperda cells, such as Sf9 cells.Recombinant virus that expresses zlmda33 protein is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

[0069] For protein production, the recombinant virus is used to infecthost cells, typically a cell line derived from the fall armyworm,Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni(e.g., High Five™ cells; Invitrogen, Carlsbad, Calif.). See, forexample, U.S. Pat. No. 5,300,435. Serum-free media are used to grow andmaintain the cells. Suitable media formulations are known in the art andcan be obtained from commercial suppliers. The cells are grown up froman inoculation density of approximately 2-5×10⁵ cells to a density of1-2×10⁶ cells, at which time a recombinant viral stock is added at amultiplicity of infection (MOI) of 0.1 to 10, more typically near 3.Procedures used are generally known in the art.

[0070] Other higher eukaryotic cells can also be used as hosts,including plant cells and avian cells. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.

[0071] Fungal cells, including yeast cells, can also be used within thepresent invention. Yeast species of particular interest in this regardinclude Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S.Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S.Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075.Transformed cells are selected by phenotype determined by the selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient (e.g., leucine). An exemplary vector system foruse in Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986; Cregg, U.S. Pat. No. 4,882,279; andRaymond et al., Yeast 14 11-23, 1998. Aspergillus cells can be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533. Production ofrecombinant proteins in Pichia methanolica is disclosed in U.S. Pat.Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.

[0072] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention. Techniques for transforming these hostsand expressing foreign DNA sequences cloned therein are well known inthe art (see, e.g., Sambrook et al., ibid.). When expressing a zlmda33polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm or may be directed to the periplasmic space by abacterial secretion sequence. In the former case the cells are lysed,and the zlmda33 polypeptide is recovered from the lysate. If thepolypeptide is present in the cytoplasm as insoluble granules, the cellsare lysed, and the granules are recovered and denatured using, forexample, guanidine isothiocyanate or urea. The denatured polypeptide canthen be refolded and dimerized by diluting the denaturant, such as bydialysis against a solution of urea and a combination of reduced andoxidized glutathione, followed by dialysis against a buffered salinesolution. In the latter case, the polypeptide can be recovered from theperiplasmic space in a soluble and functional form by disrupting thecells (by, for example, sonication or osmotic shock) to release thecontents of the periplasmic space and re covering the protein, therebyobviating the need for denaturation and refolding.

[0073] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. Liquid culturesare provided with sufficient aeration by conventional means, such asshaking of small flasks or sparging of fermentors.

[0074] Zlmda33 polypeptides can also be prepared through chemicalsynthesis according to methods known in the art, including exclusivesolid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. See, for example,Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid PhasePeptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, Ill.,1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford,1989. In vitro synthesis is particularly advantageous for thepreparation of smaller polypeptides.

[0075] Using methods known in the art, zlmda33 proteins can be preparedas monomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; and may or may not include an initial methionine aminoacid residue. In many cases, the structure of the final protein willresult from processing of the nascent polypeptide chain by the hostcell, thus the final sequence of a zlmda33 polypeptide produced by ahost cell will not always correspond to the full sequence encoded by theexpressed polynucleotide. Differential processing of individual chainsmay result in heterogeneity of expressed proteins.

[0076] Depending upon the intended use, the polypeptides and proteins ofthe present invention can be purified to ≧80% purity, ≧90% purity, ≧95%purity, or to a pharmaceutically pure state, that is greater than 99.9%pure with respect to contaminating macromolecules, particularly otherproteins and nucleic acids, and free of infectious and pyrogenic agents.Within certain embodiments, the polypeptide or protein is substantiallyfree of other polypeptides or proteins, particularly those of animalorigin.

[0077] Zlmda33 proteins (including chimeric polypeptides and multimericproteins) are purified by conventional protein purification methods,typically by a combination of chromatographic techniques. See, ingeneral, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988; and Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York, 1994. Proteinscomprising a polyhistidine affinity tag (typically about 6 histidineresidues) are purified by affinity chromatography on a nickel chelateresin. See, for example, Houchuli et al., Bio/Technol. 6: 1321-1325,1988. Proteins comprising a glu-glu tag can be purified byimmunoaffinity chromatography according to conventional procedures. See,for example, Grussenmeyer et al., ibid. Maltose binding protein fusionsare purified on an amylose column according to methods known in the art.

[0078] Biological activities of zlmda33 proteins can be measured invitro using cultured cells or in vivo by administering molecules of theclaimed invention to the appropriate animal model. Many such assays andmodels are known in the art. Guidance in initial assay selection isprovided by structural predictions and sequence alignments. However,even if no functional prediction is made, the activity of a protein canbe elucidated by known methods, including, for example, screening avariety of target cells for a biological response, other in vitroassays, expression in a host animal, or through the use of transgenicand/or “knockout” animals. Through the application of robotics, many invitro assays can be adapted to rapid, high-throughput screeing of alarge number of samples.

[0079] The activity of zlmda33 proteins can be measured with asilicon-based biosensor microphysiometer that measures the extracellularacidification rate or proton excretion associated with receptor bindingand subsequent physiologic cellular responses. An exemplary such deviceis the Cytosensor™ Microphysiometer manufactured by Molecular Devices,Sunnyvale, Calif.. A variety of cellular responses, such as cellproliferation, ion transport, energy production, inflammatory response,regulatory and receptor activation, and the like, can be measured bythis method. See, for example, McConnell et al., Science 257:1906-1912,1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli etal., J. Immunol. Meth. 212:49-59, 1998; and Van Liefde et al., Eur. J.Pharmacol. 346:87-95, 1998. The microphysiometer can be used forassaying adherent or non-adherent eukaryotic or prokaryotic cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including zlmda33 proteins, their agonists, and antagonists.

[0080] Assays measuring cell proliferation or differentiation are wellknown in the art. For example, assays measuring proliferation includesuch assays as chemosensitivity to neutral red dye (Cavanaugh et al.,Investigational New Drugs 8:347-354, 1990), incorporation ofradiolabeled nucleotides (as disclosed by, e.g., Raines and Ross,Methods Enzymol. 109:749-773, 1985; Wahl et al., Mol. Cell Biol.8:5016-5025, 1988; and Cook et al., Analytical Biochem. 179:1-7, 1989),incorporation of 5-bromo-2′-deoxyuridine (BrdU) in the DNA ofproliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179,1985), and use of tetrazolium salts (Mosmann, J. Immunol. Methods65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall etal., Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res.48:4827-4833, 1988). Differentiation can be assayed using suitableprecursor cells that can be induced to differentiate into a more maturephenotype. Assays measuring differentiation include, for example,measuring cell-surface markers associated with stage-specific expressionof a tissue, enzymatic activity, functional activity or morphologicalchanges (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation57:63-75, 1994; and Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses,161-171, 1989). Effects of a protein on tumor cell growth and metastasiscan be analyzed using the Lewis lung carcinoma model, for example asdescribed by Cao et al., J. Exp. Med. 182:2069-2077, 1995. Activity of aprotein on cells of neural origin can be analyzed using assays thatmeasure effects on neurite growth as disclosed below.

[0081] Zlmda33 activity may also be detected using assays designed tomeasure zlmda33 modulation of production of one or more additionalgrowth factors or other macromolecules. Such assays include those fordetermining the presence of hepatocyte growth factor (HGF), epidermalgrowth factor (EGF), transforming growth factor alpha (TGFα),interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF),angiogenin, and other macromolecules. Assays of IL-1 activity include,for example, gel-shift assays for NF-κB activation, Thr-669 kinaseactivity assays, and IL-8 promoter activation assays. See, Mitcham etal., J. Biol. Chem. 271:5777-5783, 1996. Suitable assays includemitogenesis assays, receptor-binding assays, competition binding assays,immunological assays (e.g., ELISA), and other formats known in the art.Metalloprotease secretion is measured from treated primary human dermalfibroblasts, synoviocytes and chondrocytes. The relative levels ofcollagenase, gelatinase and stromalysin produced in response toculturing in the presence of a zlmda33 protein is measured usingzymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-106,1990). Procollagen/collagen synthesis by dermal fibroblasts andchondrocytes in response to a test protein is measured using ³H-prolineincorporation into nascent secreted collagen. ³H-labeled collagen isvisualized by SDS-PAGE followed by autoradiography (Unemori and Amento,J. Biol. Chem. 265: 10681-10685, 1990). Glycosaminoglycan (GAG)secretion from dermal fibroblasts and chondrocytes is measured using a1,9-dimethylmethylene blue dye binding assay (Farndale et al., Biochim.Biophys. Acta 883:173-177, 1986). Inhibition of cytokine activity isassayed by including zlmda33 with one or more cytokines known to beactive in a given assay. Collagen and GAG assays, for example, arecarried out in the presence of IL-1β or TGF-β to examine the ability ofzlmda33 protein to modify the established responses to these cytokines.

[0082] Monocyte activation assays are carried out (1) to look for theability of zlmda33 proteins to modulate monocyte activation, includingattachment-induced or endotoxin-induced monocyte activation (Fuhlbriggeet al., J. Immunol. 138: 3799-3802, 1987). IL-1β and TNFα levelsproduced in response to activation are measured by ELISA (Biosource,Inc. Camarillo, Calif.). Monocyte/macrophage cells, by virtue of CD14(LPS receptor), are exquisitely sensitive to endotoxin, and proteinswith moderate levels of endotoxin-like activity will activate thesecells.

[0083] In vitro assays for pro- and anti-inflammatory activity are knownin the art. Exemplary activity assays include mitogenesis assays inwhich IL-1 responsive cells (e.g., the D10.N4.M murine T cell line) areincubated in the presence of IL-1 or a test protein for 72 hours at 37°C. in a 5% CO₂ atmosphere. IL-2 (and optionally IL-4) is added to theculture medium to enhance sensitivity and specificity of the assay.³H-thymidine is then added, and incubation is continued for six hours.The amount of label incorporated is indicative of agonist activity. See,Hopkins and Humphreys, J. Immunol. Methods 120:271-276, 1989; Greenfederet al., J. Biol. Chem. 270:22460-22466, 1995. Stimulation of cellproliferation can also be measured using thymocytes cultured in a testprotein in combination with phytohemagglutinin. IL-1 is used as acontrol. Proliferation is detected as ³H-thymidine incorporation ormetabolic breakdown of (MTT) (Mosman, ibid.).

[0084] Hematopoietic activity of zlmda33 proteins can be assayed onvarious hematopoietic cells in culture. Such assays include primary bonemarrow colony assays and later stage lineage-restricted colony assays,which are known in the art (e.g., Holly et al., WIPO Publication WO95/21920). Marrow cells plated on a suitable semi-solid medium (e.g.,50% methylcellulose containing 15% fetal bovine serum, 10% bovine serumalbumin, and 0.6% PSN antibiotic mix) are incubated in the presence oftest polypeptide, then examined microscopically for colony formation.Known hematopoietic factors are used as controls. Mitogenic activity ofzlmda33 polypeptides on hematopoietic cell lines can be measured asdisclosed above.

[0085] Cell migration is assayed essentially as disclosed by Kähler etal. (Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939,1997). A protein is considered to be chemotactic if it induces migrationof cells from an area of low protein concentration to an area of highprotein concentration. A typical assay is performed using modifiedBoyden chambers with a polystryrene membrane separating the two chambers(Transwell®; Coming Costar® Corp.). The test sample, diluted in mediumcontaining 1% BSA, is added to the lower chamber of a 24-well platecontaining Transwells. Cells are then placed on the Transwell insertthat has been pretreated with 0.2% gelatin. Cell migration is measuredafter 4 hours of incubation at 37° C. Non-migrating cells are wiped offthe top of the Transwell membrane, and cells attached to the lower faceof the membrane are fixed and stained with 0.1% crystal violet. Stainedcells are then extracted with 10% acetic acid and absorbance is measuredat 600 nm. Migration is then calculated from a standard calibrationcurve.

[0086] Cell migration can also be measured using the matrigel method ofGrant et al. (“Angiogenesis as a component of epithelial-mesenchymalinteractions” in Goldberg and Rosen, Epithelial-Mesenchymal Interactionin Cancer, Birkhäuser Verlag, 1995, 235-248; Baatout, AnticancerResearch 17:451-456, 1997).

[0087] Cell adhesion activity is assayed essentially as disclosed byLaFleur et al. (J. Biol. Chem. 272:32798-32803, 1997). Briefly,microtiter plates are coated with the test protein, non-specific sitesare blocked with BSA, and cells (such as smooth muscle cells,leukocytes, or endothelial cells) are plated at a density ofapproximately 10⁴-10⁵ cells/well. The wells are incubated at 37° C.(typically for about 60 minutes), then non-adherent cells are removed bygentle washing. Adhered cells are quantitated by conventional methods(e.g., by staining with crystal violet, lysing the cells, anddetermining the optical density of the lysate). Control wells are coatedwith a known adhesive protein, such as fibronectin or vitronectin.

[0088] Other metabolic effects of zlmda33 proteins can be measured byculturing target cells in the presence and absence of a protein andobserving changes in adipogenesis, gluconeogenesis, glycogenolysis,lipogenesis, glucose uptake, or the like. Suitable assays are known inthe art.

[0089] Proteins can be assayed for the ability to modulate axon guidanceand growth. Suitable assays that detect changes in neuron growthpatterns include, for example, those disclosed in Hastings, WIPOPublication WO 97/29189 and Walter et al., Development 101:685-696,1987. Assays to measure the effects on neuron growth are well known inthe art. For example, the C assay (e.g., Raper and Kapfhammer, Neuron4:21-29, 1990 and Luo et al., Cell 75:217-227, 1993) can be used todetermine collapsing activity of a protein of interest on growingneurons. Other methods that can assess protein-induced inhibition ofneurite extension or divert such extension are also known. See, Goodman,Annu. Rev. Neurosci. 19:341-377, 1996. Conditioned media from cellsexpressing a protein of interest, or aggregates of such cells, can byplaced in a gel matrix near suitable neural cells, such as dorsal rootganglia (DRG) or sympathetic ganglia explants, which have beenco-cultured with nerve growth factor. Compared to control cells,protein-induced changes in neuron growth can be measured (as disclosedby, for example, Messersmith et al., Neuron 14:949-959, 1995 and Puschelet al., Neuron 14:941-948, 1995). Neurite outgrowth can be measuredusing neuronal cell suspensions grown in the presence of molecules ofthe present invention. See, for example, O'Shea et al., Neuron7:231-237, 1991 and DeFreitas et al., Neuron 15:333-343, 1995.

[0090] Assays for angiogenic activity are also known in the art. Forexample, the effect of a protein of interest on primordial endothelialcells in angiogenesis can be assayed in the chick chorioallantoicmembrane angiogenesis assay (Leung, Science 246:1306-1309, 1989;Ferrara, Ann. NY Acad. Sci. 752:246-256, 1995). Briefly, a small windowis cut into the shell of an eight-day old fertilized egg, and a testsubstance is applied to the chorioallantoic membrane. After 72 hours,the membrane is examined for neovascularization. Other suitable assaysinclude microinjection of early stage quail (Coturnix coturnix japonica)embryos as disclosed by Drake et al. (Proc. Natl. Acad. Sci. USA92:7657-7661, 1995); the rodent model of corneal neovascularizationdisclosed by Muthukkaruppan and Auerbach (Science 205:1416-1418, 1979),wherein a test substance is inserted into a pocket in the cornea of aninbred mouse; and the hampster cheek pouch assay (Hockel et al., Arch.Surg. 128:423-429, 1993). Induction of vascular permeability, which isindicative of angiogenic activity, is measured in assays designed todetect leakage of protein from the vasculature of a test animal (e.g.,mouse or guinea pig) after administration of a test compound (Miles andMiles, J. Physiol. 118:228-257, 1952; Feng et al., J. Exp. Med.183:1981-1986, 1996). In vitro assays for angiogenic activity includethe tridimensional collagen gel matrix model (Pepper et al. Biochem.Biophys. Res. Comm. 189:824-831, 1992 and Ferrara et al., Ann. NY Acad.Sci. 732:246-256, 1995), which measures the formation of tube-likestructures by microvascular endothelial cells; and matrigel models(Grant et al., “Angiogenesis as a component of epithelial-mesenchymalinteractions” in Goldberg and Rosen, Epithelial-Mesenchymal Interactionin Cancer, Birkhäuser Verlag, 1995, 235-248; Baatout, AnticancerResearch 17:451-456, 1997), which are used to determine effects on cellmigration and tube formation by endothelial cells seeded in matrigel, abasement membrane extract enriched in laminin. It is preferred to carryout angiogenesis assays in the presence and absence of vascularendothelial growth factor (VEGF) to assess possible combinatorialeffects. It is also preferred to use VEGF as a control within in vivoassays.

[0091] Receptor binding can be measured by the competition bindingmethod of Labriola-Tompkins et al., Proc. Natl. Acad. Sci. USA88:11182-11186, 1991. In an exemplary assay for IL-1 receptor binding,membranes pepared from EL-4 thymoma cells (Paganelli et al., J. Immunol.138:2249-2253, 1987) are incubated in the presence of the test proteinfor 30 minutes at 37° C. Labeled IL-1α or IL-1β is then added and theincubation is continued for 60 minutes. The assay is terminated bymembrane filtration. The amount of bound label is determined byconventional means (e.g., γ counter). In an alternative assay, theability of a test protein to compete with labeled IL-1 for binding tocultured human dermal fibroblasts is measured according to the method ofDower et al. (Nature 324:266-268, 1986). Briefly, cells are incubated ina round-bottomed, 96-well plate in a suitable culture medium (e.g., RPMI1640 containing 1% BSA, 0.1% Na azide, and 20 mM HEPES pH 7.4) at 8° C.on a rocker platform in the presence of labeled IL-1. Variousconcentrations of test protein are added. After the incubation(typically about two hours), cells are separated from unbound label bycentrifuging 60-μl aliquots through 200 μl of phthalate oils in 400-μlpolyethylene centrifuge tubes and excising the tips of the tubes with arazor blade as disclosed by Segal and Hurwitz, J. Immunol.118:1338-1347, 1977. Receptor binding assays for other cell types areknown in the art. See, for example, Bowen-Pope and Ross, MethodsEnzymol. 109:69-100, 1985.

[0092] Receptor binding can also be measured using immobilized receptorsor ligand-binding receptor fragments. For example, an immobilizedreceptor can be exposed to its labeled ligand and unlabeled testprotein, whereby a reduction in labeled ligand binding compared to acontrol is indicative of receptor-binding activity in the test protein.Within another format, a receptor or ligand-binding receptor fragment isimmobilized on a biosensor (e.g., BIACore™, Pharmacia Biosensor,Piscataway, N.J.) and binding is determined. Antagonists of the nativeligand will exhibit receptor binding but will exhibit essentially noactivity in appropriate activity assays or will reduce theligand-mediated response when combined with the native ligand. In viewof the low level of receptor occupancy required to produce a response tosome ligands (e.g., IL-1), a large excess of antagonist (typically a 10-to 1000-fold molar excess) may be necessary to neutralize ligandactivity.

[0093] Receptor activation can be detected in target cells by: (1)measurement of adenylate cyclase activity (Salomon et al., Anal.Biochem. 58:541-48, 1974; Alvarez and Daniels, Anal. Biochem.187:98-103, 1990); (2) measurement of change in intracellular cAMPlevels using conventional radioimmunoassay methods (Steiner et al., J.Biol. Chem. 247:1106-13, 1972; Harper and Brooker, J. Cyc. Nucl. Res.1:207-18, 1975); or (3) through use of a cAMP scintillation proximityassay (SPA) method (such as available from Amersham Corp., ArlingtonHeights, Ill.).

[0094] Proteins can be tested for serine protease activity or proteinaseinhibitory activity using conventional assays. Substrate cleavage isconveniently assayed using a tetrapeptide that mimics the cleavage siteof the natural substrate and which is linked, via a peptide bond, to acarboxyl-terminal para-nitro-anilide (pNA) group. The proteasehydrolyzes the bond between the fourth amino acid residue and the pNAgroup, causing the pNA group to undergo a dramatic increase inabsorbance at 405 nm. Suitable substrates can be synthesized accordingto known methods or obtained from commercial suppliers. Inhibitoryactivity is measured by adding a test sample to a reaction mixturecontaining enzyme and substrate, and comparing the observed enzymeactivity to a control (without the test sample). A variety of suchassays are known in the art, including assays measuring inhibition oftrypsin, chymotrypsin, plasmin, cathepsin G, and human leukocyteelastase. See, for example, Petersen et al., Eur. J. Biochem.235:310-316, 1996. In a typical procedure, the inhibitory activity of atest compound is measured by incubating the test compound with theproteinase, then adding an appropriate substrate, typically achromogenic peptide substrate. See, for example, Norris et al. (Biol.Chem. Hoppe-Seyler 371:37-42, 1990). Various concentrations of theinhibitor are incubated in the presence of trypsin, plasmin, and plasmakallikrein in a low-salt buffer at pH 7.4, 25° C. After 30 minutes, theresidual enzymatic activity is measured by the addition of a chromogenicsubstrate (e.g., S2251 (D-Val-Leu-Lys-Nan) or S2302 (D-Pro-Phe-Arg-Nan),available from Kabi, Stockholm, Sweden) and a 30-minute incubation.Inhibition of enzyme activity is indicated by a decrease in absorbanceat 405 nm or fluorescence Em at 460 nm. From the results, the apparentinhibition constant K_(i) is calculated. When a serine protease isprepared as an active precursor, it is activated by cleavage with asuitable protease (e.g., furin (Steiner et al., J. Biol. Chem.267:23435-23438, 1992)) prior to assay. Assays of this type are wellknown in the art. See, for example, Lottenberg et al., ThrombosisResearch 28:313-332, 1982; Cho et al., Biochem. 23:644-650, 1984; Fosteret al., Biochem. 26:7003-7011, 1987). The inhibition of coagulationfactors (e.g., factor VIIa, factor Xa) can be measured using chromogenicsubstrates or in conventional coagulation assays (e.g., clotting time ofnormal human plasma; Dennis et al., J. Biol. Chem. 270:25411-25417,1995).

[0095] Blood coagulation and chromogenic assays, which can be used todetect both procoagulant, anticoagulant, and thrombolytic activities,are known in the art. For example, pro- and anticoagulant activities canbe measured in a one-stage clotting assay using platelet-poor orfactor-deficient plasma (Levy and Edgington, J. Exp. Med. 151:1232-1243,1980; Schwartz et al., J. Clin. Invest. 67:1650-1658, 1981). Asdisclosed by Anderson et al. (Proc. Natl. Acad. Sci. USA 96:11189-11193,1999), the effect of a test compound on platelet activation can bedetermined by a change in turbidity, and the procoagulant activity ofactivated platelets can be determined in a phospholipid-dependentcoagulation assay. Activation of thrombin can be determined byhydrolysis of peptide p-nitroanilide substrates as disclosed byLottenberg et al. (Thrombosis Res. 28:313-332, 1982). Otherprocoagulant, anticoagulant, and thrombolytic activities can be measuredusing appropriate chromogenic substrates, a variety of which areavailable from commercial suppliers. See, for example, Kettner and Shaw,Methods Enzymol. 80:826-842, 1981.

[0096] Anti-microbial activity of proteins is evaluated by techniquesthat are known in the art. For example, anti-microbial activity can beassayed by evaluating the sensitivity of microbial cell cultures to testagents and by evaluating the protective effect of test agents oninfected mice. See, for example, Musiek et al., Antimicrob. AgentsChemothr. 3:40, 1973. Antiviral activity can also be assessed byprotection of mammalian cell cultures. Known techniques for evaluatinganti-microbial activity include, for example, Barsum et al., Eur.Respir. J. 8:709-714, 1995; Sandovsky-Losica et al., J. Med. Vet. Mycol(England) 28:279-287, 1990; Mehentee et al., J. Gen. Microbiol (England)135(:2181-2188, 1989; and Segal and Savage, J. Med. Vet. Mycol.24:477-479, 1986. Assays specific for anti-viral activity include, forexample, those described by Daher et al., J. Virol. 60:1068-1074, 1986.

[0097] The assays disclosed above can be modified by those skilled inthe art to detect the presence of agonists and antagonists of a selectedprotein of interest.

[0098] Expression of zlmda33 polynucleotides in animals provides modelsfor further study of the biological effects of overproduction orinhibition of protein activity in vivo. Zlmda33-encoding polynucleotidesand antisense polynucleotides can be introduced into test animals, suchas mice, using viral vectors or naked DNA, or transgenic animals can beproduced.

[0099] One in vivo approach for assaying proteins of the presentinvention utilizes viral delivery systems. Exemplary viruses for thispurpose include adenovirus, herpesvirus, retroviruses, vaccinia virus,and adeno-associated virus (AAV). Adenovirus, a double-stranded DNAvirus, is currently the best studied gene transfer vector for deliveryof heterologous nucleic acids. For review, see Becker et al., Meth. CellBiol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine4:44-53, 1997. The adenovirus system offers several advantages.Adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of mammalian cell types;and (iv) be used with many different promoters including ubiquitous,tissue specific, and regulatable promoters. Because adenoviruses arestable in the bloodstream, they can be administered by intravenousinjection.

[0100] By deleting portions of the adenovirus genome, larger inserts (upto 7 kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. In an exemplary system, theessential E1 gene is deleted from the viral vector, and the virus willnot replicate unless the E1 gene is provided by the host cell (e.g., thehuman 293 cell line). When intravenously administered to intact animals,adenovirus primarily targets the liver. If the adenoviral deliverysystem has an E1 gene deletion, the virus cannot replicate in the hostcells. However, the host's tissue (e.g., liver) will express and process(and, if a signal sequence is present, secrete) the heterologousprotein. Secreted proteins will enter the circulation in the highlyvascularized liver, and effects on the infected animal can bedetermined.

[0101] An alternative method of gene delivery comprises removing cellsfrom the body and introducing a vector into the cells as a naked DNAplasmid. The transformed cells are then re-implanted in the body. NakedDNA vectors are introduced into host cells by methods known in the art,including transfection, electroporation, microinjection, transduction,cell fusion, DEAE dextran, calcium phosphate precipitation, use of agene gun, or use of a DNA vector transporter. See, Wu et al., J. Biol.Chem. 263:14621-14624, 1988; Wu et al., J. Biol. Chem. 267:963-967,1992; and Johnston and Tang, Meth. Cell Biol. 43:353-365, 1994.

[0102] Transgenic mice, engineered to express a zlmda33 gene, and micethat exhibit a complete absence of zlmda33 gene function, referred to as“knockout mice” (Snouwaert et al., Science 257:1083, 1992), can also begenerated (Lowell et al., Nature 366:740-742, 1993). These mice can beemployed to study the zlmda33 gene and the protein encoded thereby in anin vivo system. Transgenic mice are particularly useful forinvestigating the role of zlmda33 proteins in early development in thatthey allow the identification of developmental abnormalities or blocksresulting from the over- or underexpression of a specific factor. Seealso, Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science277:48-50, 1997. Promoters for transgenic expression include promotersfrom metallothionein and albumin genes.

[0103] Inhibitory polynucleotides can be used to inhibit zlmda33 geneexpression as disclosed in more detail below to examine the effects ofsuch inhibition in vivo. Inhibitory polynucleotides can also be used toinhibit expression of zlmda33 polypeptide-encoding genes in cellculture.

[0104] Biological activities of test proteins can also be measured inanimal models by administering the test protein, by itself or incombination with other agents, including other proteins. Using suchmodels facilitates the assay of the test protein by itself or as aninhibitor or modulator of another agent, and also facilitates themeasurement of combinatorial effects of bioactive compounds.

[0105] Anti-inflammatory activity can be tested in animal models ofinflammatory disease. For example, animal models of psoriasis includethe analysis of histological alterations in adult mouse tail epidermis(Hofbauer et al, Brit. J. Dermatol. 118:85-89, 1988; Bladon et al., ArchDermatol. Res. 277:121-125, 1985). In this model, anti-psoriaticactivity is indicated by the induction of a granular layer andorthokeratosis in areas of scale between the hinges of the tailepidermis. Typically, a topical ointment comprising a test compound isapplied daily for seven consecutive days, then the animal is sacrificed,and tail skin is examined histologically. An additional model isprovided by grafting psoriatic human skin to congenitally athymic (nude)mice (Krueger et al., J. Invest. Dermatol. 64:307-312, 1975). Suchgrafts have been shown to retain the characteristic histology for up toeleven weeks. As in the mouse tail model, the test composition isapplied to the skin at predetermined intervals for a period of one toseveral weeks, at which time the animals are sacrificed and the skingrafts examined histologically. A third model has been disclosed byFretland et al. (Inflammation 14:727-739, 1990). Briefly, inflammationis induced in guinea pig epidermis by topically applying phorbol ester(phorbol-12-myristate-13-acetate; PMA), typically at ca. 2 g/ml inacetone, to one ear and vehicle to the contralateral ear. Test compoundsare applied concurrently with the PMA, or may be given orally.Histological analysis is performed at 96 hours after application of PMA.This model duplicates many symptoms of human psoriasis, including edema,inflammatory cell diapedesis and infiltration, high LTB₄ levels andepidermal proliferation.

[0106] Cerebral ischemia can be studied in a rat model as disclosed byRelton et al. (ibid.) and Loddick et al. (ibid.).

[0107] The effect of a test protein on primordial endothelial cells inangiogenesis can be assayed in the chick chorioallantoic membraneangiogenesis assay (Leung, Science 246:1306-1309, 1989; Ferrara, Ann. NYAcad. Sci. 752:246-256, 1995). Briefly, a small window is cut into theshell of an eight-day old fertilized egg, and a test substance isapplied to the chorioallantoic membrane. After 72 hours, the membrane isexamined for neovascularization. Embryo microinjection of early stagequail (Coturnix coturnix japonica) embryos can also be used (Drake etal., Proc. Natl. Acad. Sci. USA 92:7657-7661, 1995). Briefly, a solutioncontaining the protein is injected into the interstitial space betweenthe endoderm and the splanchnic mesoderm of early-stage embryos using amicropipette and micromanipulator system. After injection, embryos areplaced ventral side down on a nutrient agar medium and incubated for 7hours at 37° C. in a humidified CO₂/air mixture (10%/90%). Vasculardevelopment is assessed by microscopy of fixed, whole-mounted embryosand sections.

[0108] Stimulation of coronary collateral growth can be measured inknown animal models, including a rabbit model of peripheral limbischemia and hind limb ischemia and a pig model of chronic myocardialischemia (Ferrara et al., Endocrine Reviews 18:4-25, 1997). Testproteins are assayed in the presence and absence of VEGF and basic FGFto test for combinatorial effects. These models can be modified by theuse of adenovirus or naked DNA for gene delivery as disclosed in moredetail above, resulting in local expression of the test protein(s).

[0109] Angiogenic activity can also be tested in a rodent model ofcorneal neovascularization as disclosed by Muthukkaruppan and Auerbach,Science 205:1416-1418, 1979, wherein a test substance is inserted into apocket in the cornea of an inbred mouse. For use in this assay, proteinsare combined with a solid or semi-solid, biocompatible carrier, such asa polymer pellet. Angiogenesis is followed microscopically. Vasculargrowth into the corneal stroma can be detected in about 10 days.

[0110] Angiogenic activity can also be tested in the hampster cheekpouch assay (Höckel et al., Arch. Surg. 128:423-429, 1993). A testsubstance is injected subcutaneiously into the cheek pouch, and afterfive days the pouch is examined under low magnification to determine theextent of neovascularization. Tissue sections can also be examinedhistologically.

[0111] Induction of vascular permeability is measured in assays designedto detect leakage of protein from the vasculature of a test animal(e.g., mouse or guinea pig) after administration of a test compound(Miles and Miles, J. Physiol. 118:228-257, 1952; Feng et al., J. Exp.Med. 183:1981-1986, 1996).

[0112] Wound-healing models include the linear skin incision model ofMustoe et al. (Science 237:1333, 1987). In a typical procedure, a 6-cmincision is made in the dorsal pelt of an adult rat, then closed withwound clips. Test substances and controls (in solution, gel, or powderform) are applied before primary closure. It is preferred to limitadministration to a single application, although additional applicationscan be made on succeeding days by careful injection at several sitesunder the incision. Wound breaking strength is evaluated between 3 and21 days post wounding. In a second model, multiple, small,full-thickness excisions are made on the ear of a rabbit. The cartilagein the ear splints the wound, removing the variable of wound contractionfrom the evaluation of closure. Experimental treatments and controls areapplied. The geometry and anatomy of the wound site allow for reliablequantification of cell ingrowth and epithelial migration, as well asquantitative analysis of the biochemistry of the wounds (e.g., collagencontent). See, Mustoe et al., J. Clin. Invest. 87:694, 1991. The rabbitear model can be modified to create an ischemic wound environment, whichmore closely resembles the clinical situation (Ahn et al., Ann. Plast.Surg. 24:17, 1990). Within a third model, healing of partial-thicknessskin wounds in pigs or guinea pigs is evaluated (LeGrand et al., GrowthFactors 8:307, 1993). Experimental treatments are applied daily on orunder dressings. Seven days after wounding, granulation tissue thicknessis determined. This model is preferred for dose-response studies, as itis more quantitative than other in vivo models of wound healing. A fullthickness excision model can also be employed. Within this model, theepidermis and dermis are removed down to the panniculus carnosum inrodents or the subcutaneous fat in pigs. Experimental treatments areapplied topically on or under a dressing, and can be applied daily ifdesired. The wound closes by a combination of contraction and cellingrowth and proliferation. Measurable endpoints include time to woundclosure, histologic score, and biochemical parameters of wound tissue.Impaired wound healing models are also known in the art (e.g., Cromacket al., Surgery 113:36, 1993; Pierce et al., Proc. Natl. Acad. Sci. USA86:2229, 1989; Greenhalgh et al., Amer. J. Pathol. 136:1235, 1990).Delay or prolongation of the wound healing process can be inducedpharmacologically by treatment with steroids, irradiation of the woundsite, or by concomitant disease states (e.g., diabetes). Linearincisions or full-thickness excisions are most commonly used as theexperimental wound. Endpoints are as disclosed above for each type ofwound. Subcutaneous implants can be used to assess compounds acting inthe early stages of wound healing (Broadley et al., Lab. Invest. 61:571,1985; Sprugel et al., Amer. J. Pathol. 129: 601, 1987). Implants areprepared in a porous, relatively non-inflammatory container (e.g.,polyethylene sponges or expanded polytetrafluoroethylene implants filledwith bovine collagen) and placed subcutaneously in mice or rats. Theinterior of the implant is empty of cells, producing a “wound space”that is well-defined and separable from the preexisting tissue. Thisarrangement allows the assessment of cell influx and cell type as wellas the measurement of vasculogenesis/angiogenesis and extracellularmatrix production.

[0113] Inhibition of tumor metastasis can be assessed in mice into whichcancerous cells or tumor tissue have been introduced by implantation orinjection (e.g., Brown, Advan. Enzyme Regul. 35:293-301, 1995; Conway etal., Clin. Exp. Metastasis 14:115-124, 1996).

[0114] Effects on fibrinolysis can be measured in a rat model whereinthe enzyme batroxobin and radiolabeled fibrinogen are administered totest animals. Inhibition of fibrinogen activation by a test compound isseen as a reduction in the circulating level of the label as compared toanimals not receiving the test compound. See, Lenfors and Gustafsson,Semin. Thromb. Hemost. 22:335-342, 1996.

[0115] The polypeptides, nucleic acids and antibodies of the presentinvention may be used in diagnosis or treatment of disorders associatedwith cell loss or abnormal cell physiology (including cancer). Analysisof gene expression has shown that zlmda33 is expressed in ovarian anduterine tumors, but not in the corresponding normal tissues. Zlmda33 isthus a diagnostic marker of these tumors. Those skilled in the art willrecognize that assays can be performed on body fluids (e.g., plasma,serum, urine), tissue samples, or isolated cells. Such diagnosis willgenerally be carried out by testing a fluid or tissue sample usingconventional immunoassay methods such as enzyme-linked immunoadsorptionassays or radioimmune assays. Assays of these types are well known inthe art. See, for example, Hart et al., Biochem. 29:166-172, 1990; Ma etal., British J. Haematol. 80:431-436, 1992; and Andre et al., Clin.Chem. 38/5:758-763, 1992. In addition, zlmda33 provides a target fortherapeutic agents.

[0116] Assays for zlmda33 can be used to detect soluble protein in bodyfluids (e.g., plasma, serum, urine) or cell-associated protein inisolated cells, cell fractions (e.g., membranes), or tissue samples.General methods for collecting samples and assaying for the presence andamount of a protein are known in the art. Assays will commonly employ ananti-zlmda33 antibody or other specific binding partner (e.g., solublereceptor). The antibody or binding partner can itself be labeled,thereby directly providing a detectable signal, or a labeled secondantibody or binding partner can be used to provide the signal.

[0117] Labeled anti-zlmda33 antibodies or other binding partners may beused in vivo for imaging tumors or other sites of abnormal cellproliferation. Anti-zlmda33 antibodies or other binding partners can bedirectly or indirectly conjugated to radionuclides or other detectablemolecules, and these conjugates used for diagnostic or therapeuticapplications. For in vivo use, an anti-zlmda33 antibody or other bindingpartner can be directly or indirectly coupled to a detectable moleculeand delivered to a mammal having cells, tissues, or organs that expresszlmda33. Suitable detectable molecules include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles, electron-dense compounds, heavy metals, andthe like. These can be either directly attached to the antibody or otherbinding partner, or indirectly attached according to known methods, suchas through a chelating moiety. For indirect attachment of a detectablemolecule, the detectable molecule can be conjugated with a first memberof a complementary/anticomplementary pair, wherein the second member ofthe pair is bound to the anti-zlmda33 antibody or other binding partner.Biotin/streptavidin is an exemplary complementary/anticomplementarypair; others will be evident to those skilled in the art. The labeledcompounds described herein can be delivered intravenously,intra-arterially or intraductally, or may be introduced locally at theintended site of action.

[0118] As used herein, the term “antibodies” includes polyclonalantibodies, monoclonal antibodies, antigen-binding fragments thereofsuch as F(ab′)₂ and Fab fragments, single chain antibodies, and thelike, including genetically engineered antibodies. Non-human antibodiesmay be humanized by grafting non-human CDRs onto human framework andconstant regions, or by incorporating the entire non-human variabledomains (optionally “cloaking” them with a human-like surface byreplacement of exposed residues, wherein the result is a “veneered”antibody). In some instances, humanized antibodies may retain non-humanresidues within the human variable region framework domains to enhanceproper binding characteristics. Through humanizing antibodies,biological half-life may be increased, and the potential for adverseimmune reactions upon administration to humans is reduced. One skilledin the art can generate humanized antibodies with specific and differentconstant domains (i.e., different Ig subclasses) to facilitate orinhibit various immune functions associated with particular antibodyconstant domains. Antibodies are defined to be specifically binding ifthey bind to a zlmda33 polypeptide or protein with an affinity at least10-fold greater than the binding affinity to control (non-zlmda33)polypeptide or protein. The affinity of a monoclonal antibody can bereadily determined by one of ordinary skill in the art (see, forexample, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).

[0119] Methods for preparing polyclonal and monoclonal antibodies arewell known in the art (see for example, Hurrell, J. G. R., Ed.,Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press,Inc., Boca Raton, Fla., 1982). As would be evident to one of ordinaryskill in the art, polyclonal antibodies can be generated from a varietyof warm-blooded animals such as horses, cows, goats, sheep, dogs,chickens, rabbits, mice, and rats. The immunogenicity of a zlmda33polypeptide may be increased through the use of an adjuvant such as alum(aluminum hydroxide) or Freund's complete or incomplete adjuvant.Polypeptides useful for immunization also include fusion polypeptides,such as fusions of a zlmda33 polypeptide or a portion thereof with animmunoglobulin polypeptide or with maltose binding protein. If thepolypeptide is “hapten-like”, it may be advantageously joined or linkedto a macromolecular carrier (such as keyhole limpet hemocyanin (KLH),bovine serum albumin (BSA) or tetanus toxoid) for immunization.

[0120] Alternative techniques for generating or selecting antibodiesinclude in vitro exposure of lymphocytes to zlmda33 polypeptides, andselection of antibody display libraries in phage or similar vectors(e.g., through the use of immobilized or labeled zlmda33 polypeptide).Human antibodies can be produced in transgenic, non-human animals thathave been engineered to contain human immunoglobulin genes as disclosedin WIPO Publication WO 98/24893. It is preferred that the endogenousimmunoglobulin genes in these animals be inactivated or eliminated, suchas by homologous recombination.

[0121] A variety of assays known to those skilled in the art can beutilized to detect antibodies that specifically bind to zlmda33polypeptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include concurrentimmunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations,enzyme-linked immunosorbent assays (ELISA), dot blot assays, Westernblot assays, inhibition or competition assays, and sandwich assays.

[0122] In addition to the diagnostic and therapeutic uses disclosedabove, anti-zlmda33 antibodies can be used for affinity purification ofthe protein, for immunolocalization within whole animals or tissuesections, for immunohistochemistry, and as antagonists to block proteinactivity in vitro and in vivo. Antibodies to zlmda33 can also be used inanalytical methods employing fluorescence-activated cell sorting (FACS),for screening expression libraries, and for generating anti-idiotypicantibodies.

[0123] For pharmaceutical use, zlmda33 proteins, anti-zlmda33antibodies, and other bioactive compounds are formulated for topical orparenteral, particularly intravenous or subcutaneous, delivery accordingto conventional methods. In general, pharmaceutical formulations willinclude a zlmda33 polypeptide, antibody, or other compound incombination with a pharmaceutically acceptable vehicle, such as saline,buffered saline, 5% dextrose in water, or the like. Formulations mayfurther include one or more excipients, preservatives, solubilizers,buffering agents, albumin to prevent protein loss on vial surfaces, etc.Methods of formulation are well known in the art and are disclosed, forexample, in Remington: The Science and Practice of Pharmacy, Gennaro,ed., Mack Publishing Co., Easton, Pa., 19th ed., 1995. Zlmda33 willcommonly be used in a concentration of about 10 to 100 μg/ml of totalvolume, although concentrations in the range of 1 ng/ml to 1000 μg/mlmay be used. For topical application the protein will be applied in therange of 0.1-10 μg/cm² of surface area. The exact dose will bedetermined by the clinician according to accepted standards, taking intoaccount the nature and severity of the condition to be treated, patienttraits, etc. Determination of dose is within the level of ordinary skillin the art. Dosing is daily or intermittently over the period oftreatment. Intravenous administration will be by bolus injection orinfusion over a typical period of one to several hours. Sustainedrelease formulations can also be employed.

[0124] Within the laboratory research field, zlmda33 proteins can alsobe used as molecular weight standards or as reagents in assays fordetermining circulating levels of the protein, such as in the diagnosisof disorders characterized by over- or under-production of zlmda33protein or in the analysis of cell phenotype.

[0125] Polynucleotides and polypeptides of the present invention willadditionally find use as educational tools within laboratory practicumkits for courses related to genetics, molecular biology, proteinchemistry, and antibody production and analysis. Due to their uniquepolynucleotide and polypeptide sequences, molecules of zlmda33 can beused as standards or as “unknowns” for testing purposes. For example,zlmda33 polynucleotides can be used as an aid in teaching a student howto prepare expression constructs for bacterial, viral, and/or mammalianexpression, including fusion constructs, wherein zlmda33 is the gene tobe expressed; for experimentally determining the restrictionendonuclease cleavage sites of the polynucleotides (which can bedetermined from the sequence using conventional computer software, suchas MapDraw™ (DNASTAR, Madison, Wis.)); determining mRNA and DNAlocalization of zlmda33 polynucleotides in tissues (e.g., by Northernblotting, Southern blotting, or polymerase chain reaction); and foridentifying related polynucleotides and polypeptides by nucleic acidhybridization.

[0126] Zlmda33 polypeptides can be used educationally as aids to teachpreparation of antibodies; identification of proteins by Westernblotting; protein purification; determination of the weight of expressedzlmda33 polypeptides as a ratio to total protein expressed;identification of peptide cleavage sites; coupling amino and carboxylterminal tags; amino acid sequence analysis; as well as, but not limitedto, monitoring biological activities of both the native and taggedprotein (i.e., receptor binding, signal transduction, proliferation, anddifferentiation) in vitro and in vivo. Zlmda33 polypeptides can also beused to teach analytical skills such as mass spectrometry, circulardichroism to determine conformation, x-ray crystallography to determinethe three-dimensional structure in atomic detail, nuclear magneticresonance spectroscopy to reveal the structure of proteins in solution,and the like. For example, a kit containing a zlmda33 polypeptide can begiven to the student to analyze. Since the amino acid sequence would beknown by the instructor, the polypeptide can be given to the student asa test to determine the skills or develop the skills of the student, andthe instructor would then know whether or not the student has correctlyanalyzed the polypeptide. Since every polypeptide is unique, theeducational utility of zlmda33 would be unique unto itself.

[0127] Zlmda33 proteins can also be used to identify inhibitors of theiractivity. Test compounds are added to the assays disclosed above toidentify compounds that inhibit the activity of zlmda33 protein. Inaddition to those assays disclosed above, cellular proteins that bind toand interact with zlmda33 can be identified by, for example, screeningcDNA libraries in a yeast two-hybrid system (Fields and Song, Nature340:245-246, 1989; Gyuris et. al., Cell 75:791-803, 1993; and Li andFields, FASEB J. 7:957-963, 1993). Briefly, the yeast two-hybrid systemallows the detection of protein-protein interactions through the use oftranscriptional activators, which are modular in nature. A known gene iscloned into a “bait” vector, from which it is expressed as a fusionprotein the binding domain of a transcriptional activator. The cDNAlibrary is cloned into a second (“prey”) vector for expression of fusionproteins comprising the activation domain of the transcriptionalactivator. When proteins expressed from the two vectors interact, afunctional transcriptional activator is produced, allowing expression ofa selectable marker and consequent growth of the host cell. Vectors andother reagents for yeast two-hybrid systems are available fromcommercial suppliers (e.g., Clontech Laboratories, Inc., Palo Alto,Calif. and Invitrogen, Carlsbad, Calif.). Proteins that bind to zlmda33provide additional targets through which zlmda activity can bemodulated.

[0128] The polynucleotides of the present invention can be used indiagnostic applications. For example, the zlmda33 gene, a probecomprising zlmda33 DNA or RNA, or a subsequence thereof can be used todetermine the presence of mutations at or near the zlmda33 locus.Detectable chromosomal aberrations at the zlmda33 gene locus include,but are not limited to, aneuploidy, gene copy number changes,insertions, deletions, restriction site changes, and rearrangements.These aberrations can occur within the coding sequence, within introns,or within flanking sequences, including upstream promoter and regulatoryregions, and may be manifested as physical alterations within a codingsequence or changes in gene expression level. Analytical probes willgenerally be at least 20 nucleotides in length, although somewhatshorter probes (14-17 nucleotides) can be used. PCR primers are at least5 nucleotides in length, preferably 15 or more nt, more preferably 20-30nt. Short polynucleotides can be used when a small region of the gene istargetted for analysis.

[0129] For gross analysis of genes, a polynucleotide probe may comprisean entire exon or more. Probes will generally comprise a polynucleotidelinked to a signal-generating moiety such as a radionucleotide. Ingeneral, these diagnostic methods comprise the steps of (a) obtaining agenetic sample from a patient; (b) incubating the genetic sample with apolynucleotide probe or primer as disclosed above, under conditionswherein the polynucleotide will hybridize to complementarypolynucleotide sequence, to produce a first reaction product; and (c)comparing the first reaction product to a control reaction product. Adifference between the first reaction product and the control reactionproduct is indicative of a genetic abnormality in the patient. Geneticsamples for use within the present invention include genomic DNA, cDNA,and RNA. The polynucleotide probe or primer can be RNA or DNA, and willcomprise a portion of SEQ ID NO: 1, the complement of SEQ ID NO: 1, oran RNA equivalent thereof. Suitable assay methods in this regard includemolecular genetic techniques known to those in the art, such asrestriction fragment length polymorphism (RFLP) analysis, short tandemrepeat (STR) analysis employing PCR techniques, ligation chain reaction(Barany, PCR Methods and Applications 1:5-16, 1991), ribonucleaseprotection assays, and other genetic linkage analysis techniques knownin the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; A. J.Marian, Chest 108:255-65, 1995). Ribonuclease protection assays (see,e.g., Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNAprobe to a patient RNA sample, after which the reaction product (RNA-RNAhybrid) is exposed to RNase. Hybridized regions of the RNA are protectedfrom digestion. Within PCR assays, a patient genetic sample is incubatedwith a pair of polynucleotide primers, and the region between theprimers is amplified and recovered. Changes in size or amount ofrecovered product are indicative of mutations in the patient. AnotherPCR-based technique that can be employed is single strand conformationalpolymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications1:34-38, 1991).

[0130] Those skilled in the art will also recognize that the diagnostictechniques disclosed above can be applied to detect expression ofzlmda33 (production of mRNA) within a cell or tissue, including a cellor tissue obtained from a patient. As disclosed above, a genetic sampleis analyzed using probes or primers comprising zlmda33 DNA or RNA, or asubsequence thereof.

[0131] Sequence tagged sites (STSs) can also be used independently forchromosomal localization. An STS is a DNA sequence that is unique in thehuman genome and can be used as a reference point for a particularchromosome or region of a chromosome. An STS is defined by a pair ofoligonucleotide primers that are used in a polymerase chain reaction tospecifically detect this site in the presence of all other genomicsequences. Since STSs are based solely on DNA sequence they can becompletely described within an electronic database, for example,Database of Sequence Tagged Sites (dbSTS), GenBank (National Center forBiological Information, National Institutes of Health, Bethesda, Md.http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequenceof interest for the mapping data contained within these short genomiclandmark STS sequences.

[0132] The zlmda33 gene maps to human chromosome 8 at 8p23.1-p22. Adescription of this region of the genome is available from the OMIM™Database, Johns Hopkins University, 2000(http://www.ncbi.nlm.nih.gov/entrez/querv.fcgi?db=OMIM). The zlmda33gene is located in a region of chromosome 8 that has been found to bedeleted in many human epithelial malignancies, including liver cancer,and is involved in a translocation event associated with hepatitis Bvirus integration, an event that has been implicated in liveroncogenesis. It is believed that one or more tumor suppressor genes maybe located in this region of chromosome 8. See, Pineau et al., J. Virol.70:7280-7284, 1996.

[0133] Inhibitors of zlmda33 activity (zlmda33 antagonists) includeanti-zlmda33 antibodies, inactive receptor-binding fragments of zlmda33polypeptides, soluble zlmda33 receptors, and other peptidic andnon-peptidic agents (including inhibitory polynucleotides and smallmolecule inhibitors). Such antagonists can be used to block the effectsof zlmda33 on cells or tissues. Antagonists are formulated forpharmaceutical use as generally disclosed above, taking into account theprecise chemical and physical nature of the inhibitor and the conditionto be treated. The relevant determinations are within the level ofordinary skill in the formulation art.

[0134] Polynucleotides encoding zlmda33 polypeptides and inhibitorypolynucleotides are useful within gene therapy applications where it isdesired to increase or inhibit zlmda33 activity. If a mammal has amutated or absent zlmda33 gene, a zlmda33 gene can be introduced intothe cells of the mammal. In one embodiment, a gene encoding a zlmda33polypeptide is introduced in vivo in a viral vector. Such vectorsinclude an attenuated or defective DNA virus, such as, but not limitedto, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus(EBV), adenovirus, adeno-associated virus (AAV), and the like. Defectiveviruses, which entirely or almost entirely lack viral genes, arepreferred. A defective virus is not infective after introduction into acell. Use of defective viral vectors allows for administration to cellsin a specific, localized area, without concern that the vector caninfect other cells. Examples of particular vectors include, but are notlimited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt etal., Molec. Cell. Neurosci. 2:320-330, 1991); an attenuated adenovirusvector, such as the vector described by Stratford-Perricaudet et al., J.Clin. Invest. 90:626-630, 1992; and a defective adeno-associated virusvector (Samulski et al., J. Virol. 61:3096-3101, 1987; Samulski et al.,J. Virol. 63:3822-3888, 1989). Within another embodiment, a zlmda33 genecan be introduced in a retroviral vector as described, for example, byAnderson et al., U.S. Pat. No. 5,399,346; Mann et al. Cell 33:153, 1983;Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No.4,980,289; Markowitz et al., J. Virol. 35 62:1120, 1988; Temin et al.,U.S. Pat. No. 5,124,263; Dougherty et al., WIPO Publication WO 95/07358;and Kuo et al., Blood 82:845, 1993. Alternatively, the vector can beintroduced by liposome-mediated transfection (“lipofection”). Syntheticcationic lipids can be used to prepare liposomes for in vivotransfection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417,1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-8031, 1988). Theuse of lipofection to introduce exogenous polynucleotides into specificorgans in vivo has certain practical advantages, including moleculartargeting of liposomes to specific cells. Directing transfection toparticular cell types is particularly advantageous in a tissue withcellular heterogeneity, such as the pancreas, liver, kidney, and brain.Lipids may be chemically coupled to other molecules for the purpose oftargeting. Peptidic and non-peptidic molecules can be coupled toliposomes chemically. Within another embodiment, cells are removed fromthe body, a vector is introduced into the cells as a naked DNA plasmid,and the transformed cells are re-implanted into the body as disclosedabove.

[0135] Inhibitory polynucleotides can be used to inhibit expression ofzlmda33. Inhibitory polynucleotides include antisense polynucleotides,ribozymes, and external guide sequences.

[0136] Antisense polynucleotides can be used to inhibit zlmda33 genetranscription. Polynucleotides that are complementary to a segment of azlmda33-encoding polynucleotide (e.g., a polynucleotide as set forth inSEQ ID NO: 1) are designed to bind to zlmda33-encoding mRNA and toinhibit translation of such mRNA. Antisense polynucleotides can betargetted to specific tissues using a gene therapy approach withspecific vectors and/or promoters, such as viral delivery systems.

[0137] Ribozymes can also be used as zlmda33 antagonists. Ribozymes areRNA molecules that contain a catalytic center and a target RNA bindingportion. The term includes RNA enzymes, self-splicing RNAs,self-cleaving RNAs, and nucleic acid molecules that perform thesecatalytic functions. A ribozyme selectively binds to a target RNAmolecule through complementary base pairing, bringing the catalyticcenter into close proximity with the target sequence. The ribozyme thencleaves the target RNA and is released, after which it is able to bindand cleave additional molecules. A nucleic acid molecule that encodes aribozyme is termed a “ribozyme gene.” Ribozymes can be designed toexpress endonuclease activity that is directed to a certain targetsequence in a mRNA molecule (see, for example, Draper and Macejak, U.S.Pat. No. 5,496,698, McSwiggen, U.S. Pat. No. 5,525,468, Chowrira andMcSwiggen, U.S. Pat. No. 5,631,359, and Robertson and Goldberg, U.S.Pat. No. 5,225,337). An expression vector can be constructed in which aregulatory element is operably linked to a nucleotide sequence thatencodes a ribozyme.

[0138] In another approach, expression vectors can be constructed inwhich a regulatory element directs the production of RNA transcriptscapable of promoting RNase P-mediated cleavage of mRNA molecules thatencode a zlmda33 polypeptide. An external guide sequence is constructedfor directing the endogenous ribozyme, RNase P, to a particular speciesof intracellular mRNA, which is subsequently cleaved by the cellularribozyme (see, for example, Altman et al., U.S. Pat. No. 5,168,053; Yuanet al., Science 263:1269, 1994; Pace et al., WIPO Publication No. WO96/18733; George et al., WIPO Publication No. WO 96/21731; and Werner etal., WIPO Publication No. WO 97/33991). An external guide sequencegenerally comprises a ten- to fifteen-nucleotide sequence complementaryto zlmda33 mRNA, and a 3′-NCCA nucleotide sequence, wherein N ispreferably a purine. The external guide sequence transcripts bind to thetargeted mRNA species by the formation of base pairs between the mRNAand the complementary external guide sequences, thus promoting cleavageof mRNA by RNase P at the nucleotide located at the 5′-side of thebase-paired region.

[0139] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1

[0140] Recombinant zlmda33 is produced in E. coli using a His₆tag/maltose binding protein (MBP) double affinity fusion system asgenerally disclosed by Pryor and Leiting, Prot. Expr. Pur. 10:309-319,1997. A thrombin cleavage site is placed at the junction between theaffinity tag and zlmda33 sequences.

[0141] The fusion construct is assembled in the vector pTAP98, whichcomprises sequences for replication and selection in E. coli and yeast,the E. coli tac promoter, and a unique SmaI site just downstream of theMBP-His₆-thrombin site coding sequences. The zlmda33 cDNA (SEQ ID NO: 1)is amplified by PCR using primers each comprising 40 bp of sequencehomologous to vector sequence and 25 bp of sequence that anneals to thecDNA. The reaction is run using Taq DNA polymerase (Boehringer Mannheim,Indianapolis, Ind.) for 30 cycles of 94° C., 30 seconds; 60° C., 60seconds; and 72° C., 60 seconds. One microgram of the resulting fragmentis mixed with 100 ng of SmaI-cut pTAP98, and the mixture is transformedinto yeast to assemble the vector by homologous recombination (Oldenburget al., Nucl. Acids. Res. 25:451-452, 1997). Ura⁺ transformants areselected.

[0142] Plasmid DNA is prepared from yeast transformants and transformedinto E. coli MC1061. Pooled plasmid DNA is then prepared from the MC1061transformants by the miniprep method after scraping an entire plate.Plasmid DNA is analyzed by restriction digestion.

[0143]E. coli strain BL21 is used for expression of zlmda33. Cells aretransformed by electroporation and grown on minimal glucose platescontaining casamino acids and ampicillin.

[0144] Protein expression is analyzed by gel electrophoresis. Cells aregrown in liquid glucose media containing casamino acids and ampicillin.After one hour at 37° C., IPTG is added to a final concentration of 1mM, and the cells are grown for an additional 2-3 hours at 37° C. Cellsare disrupted using glass beads, and extracts are prepared.

Example 2

[0145] Larger scale cultures of zlmda33 transformants are prepared bythe method of Pryor and Leiting (ibid.). 100-mil cultures in minimalglucose media containing casamino acids and 100 μg/ml ampicillin aregrown at 37° C. in 500-mil baffled flasks to OD₆₀₀≈0.5. Cells areharvested by centrifugation and resuspended in 100 ml of the same mediaat room temperature. After 15 minutes, IPTG is added to 0.5 mM, andcultures are incubated at room temperature (ca. 22.5° C.) for 16 to 20hours with shaking at 125 rpm. The culture is harvested bycentrifugation, and cell pellets are stored at −70° C.

Example 3

[0146] For larger-scale protein preparation, 500-mil cultures of E. coliBL21 expressing the zlmda33-MBP-His₆ fusion protein are preparedessentially as disclosed in Example 3. Cell pellets are resuspended in100 ml of binding buffer (20 mM Tris, pH 7.58, 100 mM NaCl, 20 mMNaH₂PO₄, 0.4 mM 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride[Pefabloc® SC; Boehringer-Mannheim, Indianapolis, Ind.], 2 μg/mlLeupeptin, 2 μg/ml Aprotinin). The cells are lysed in a French press at30,000 psi, and the lysate is centrifuged at 18,000× g for 45 minutes at4° C. to clarify it. Protein concentration is estimated by gelelectrophoresis with a BSA standard.

[0147] Recombinant zlmda33 fusion protein is purified from the lysate byaffinity chromatography. Immobilized cobalt resin (Talon® metal affinityresin; Clontech Laboratories, Inc., Palo Alto, Calif.) is equilibratedin binding buffer. One ml of packed resin per 50 mg protein is combinedwith the clarified supernatant in a tube, and the tube is capped andsealed, then placed on a rocker overnight at 4° C. The resin is thenpelleted by centrifugation at 4° C. and washed three times with bindingbuffer. Protein is eluted with binding buffer containing 0.2M imidazole.The resin and elution buffer are mixed for at least one hour at 4° C.,the resin is pelleted, and the supernatant is removed. An aliquot isanalyzed by gel electrophoresis, and concentration is estimated. Amyloseresin is equilibrated in amylose binding buffer (20 mM Tris-HCl, pH 7.0,100 mM NaCl, 10 mM EDTA) and combined with the supernatant from theTalon resin at a ratio of 2 mg fusion protein per ml of resin. Bindingand washing steps are carried out as disclosed above. Protein is elutedwith amylose binding buffer containing 10 mM maltose using as small avolume as possible to minimize the need for subsequent concentration.The eluted protein is analyzed by gel electrophoresis and staining withCoomassie blue using a BSA standard, and by Western blotting using ananti-MBP antibody.

Example 4

[0148] An expression plasmid containing all or part of a polynucleotideencoding zlmda33 is constructed via homologous recombination. A fragmentof zlmda33 cDNA is isolated by PCR using primers that comprise, from 5′to 3′ end, 40 bp of flanking sequence from the vector and 17 bpcorresponding to the amino and carboxyl termini from the open readingframe of zlmda33. The resulting PCR product includes flanking regions atthe 5′ and 3′ ends corresponding to the vector sequences flanking thezlmda33 insertion point. Ten μl of the 100 μl PCR reaction mixture isrun on a 0.8% low-melting-temperature agarose (SeaPlaque GTG®; FMCBioProducts, Rockland, Me.) gel with 1×TBE buffer for analysis. Theremaining 90 μl of the reaction mixture is precipitated with theaddition of 5 μl 1 M NaCl and 250 μl of absolute ethanol.

[0149] The plasmid pZMP6, which has been cut with SmaI, is used forrecombination with the PCR fragment. Plamid pZMP6 is a mammalianexpression vector containing an expression cassette having thecytomegalovirus immediate early promoter, multiple restriction sites forinsertion of coding sequences, a stop codon, and a human growth hormoneterminator; an E. coli origin of replication; a mammalian selectablemarker expression unit comprising an SV40 promoter, enhancer and originof replication, a DHFR gene, and the SV40 terminator; and URA3 andCEN-ARS sequences required for selection and replication in S.cerevisiae. It was constructed from pZP9 (deposited at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, under Accession No. 98668) with the yeast genetic elementstaken from pRS316 (available from the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209, under AccessionNo. 77145), an internal ribosome entry site (IRES) element frompoliovirus, and a sequence encoding the extracellular domain of CD8truncated at the C-terminal end of the transmembrane domain.

[0150] One hundred microliters of competent yeast (S. cerevisiae) cellsare combined with 10 μl of the DNA preparations from above andtransferred to a 0.2-cm electroporation cuvette. The yeast/DNA mixtureis electropulsed using power supply (BioRad Laboratories, Hercules,Calif.) settings of 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To each cuvette isadded 600 μl of 1.2 M sorbitol, and the yeast is plated in two 300-μlaliquots onto two URA-D (selective media lacking uracil and containingglucose) plates and incubated at 30° C. After about 48 hours, the Ura⁺yeast transformants from a single plate are resuspended in 1 ml H₂O andspun briefly to pellet the yeast cells. The cell pellet is resuspendedin 1 ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mMTris, pH 8.0, 1 mM EDTA). Five hundred microliters of the lysis mixtureis added to an Eppendorf tube containing 300 μl acid-washed glass beadsand 200 μl phenol-chloroform, vortexed for 1 minute intervals two orthree times, and spun for 5 minutes in an Eppendorf centrifuge atmaximum speed. Three hundred microliters of the aqueous phase istransferred to a fresh tube, and the DNA is precipitated with 600 μlethanol (EtOH), followed by centrifugation for 10 minutes at 4° C. TheDNA pellet is resuspended in 10 μl H₂O.

[0151] Transformation of electrocompetent E. coli host cells (ElectromaxDH10B™ cells; obtained from Life Technologies, Inc., Gaithersburg, Md.)is done with 0.5-2 ml yeast DNA prep and 40 μl of cells. The cells areelectropulsed at 1.7 kV, 25 μF, and 400 ohms. Following electroporation,1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) is plated in 250-μl aliquots on four LB AMP plates (LB broth(Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/L Ampicillin).

[0152] Individual clones harboring the correct expression construct forzlmda33 are identified by restriction digestion to verify the presenceof the zlmda33 insert and to confirm that the various DNA sequences havebeen joined correctly to one another. The inserts of positive clones aresubjected to sequence analysis. Larger scale plasmid DNA is isolatedusing a commercially available kit (QIAGEN Plasmid Maxi Kit, Qiagen,Valencia, Calif.) according to manufacturer's instructions. The correctconstruct is designated pZMP6/zlmda33.

Example 5

[0153] CHO DG44 cells (Chasin et al., Som. Cell. Molec. Genet.12:555-666, 1986) are plated in 10-cm tissue culture dishes and allowedto grow to approximately 50% to 70% confluency overnight at 37° C., 5%CO₂, in Ham's F12/FBS media Ham's F12 medium (Life Technologies), 5%fetal bovine serum (Hyclone, Logan, Utah), 1% L-glutamine (JRHBiosciences, Lenexa, Kans.), 1% sodium pyruvate (Life Technologies)).The cells are then transfected with the plasmid zlmda33/pZMP6 byliposome-mediated transfection using a 3:1 (w/w) liposome formulation ofthe polycationic lipid2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium-trifluoroacetateand the neutral lipid dioleoyl phosphatidylethanolamine inmembrane-filetered water (Lipofectamine™ Reagent, Life Technologies), inserum free (SF) media formulation (Ham's F12, 10 mg/ml transferrin, 5mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).pZMP6/zlmda33 is diluted into 15-mi tubes to a total final volume of 640μl with SF media. 35 μl of Lipofectamine™ is mixed with 605 μl of SFmedium. The resulting mixture is added to the DNA mixture and allowed toincubate approximately 30 minutes at room temperature. Five mil of SFmedia is added to the DNA:Lipofectamine™ mixture. The cells are rinsedonce with 5 ml of SF media, aspirated, and the DNA:Lipofectamine™mixture is added. The cells are incubated at 37° C. for five hours, then6.4 ml of Ham's F12/10% FBS, 1% PSN media is added to each plate. Theplates are incubated at 37° C. overnight, and the DNA:Lipofectamine™mixture is replaced with fresh 5% FBS/Ham's media the next day. On day 3post-transfection, the cells are split into T-175 flasks in growthmedium. On day 7 postransfection, the cells are stained withFITC-anti-CD8 monoclonal antibody (Pharmingen, San Diego, Calif.)followed by anti-FITC-conjugated magnetic beads (Miltenyi Biotec). TheCD8-positive cells are separated using commercially available columns(mini-MACS columns; Miltenyi Biotec) according to the manufacturer'sdirections and put into DMEM/Ham's F12/5% FBS without nucleosides butwith 50 nM methotrexate (selection medium).

[0154] Cells are plated for subcloning at a density of 0.5, 1 and 5cells per well in 96-well dishes in selection medium and allowed to growout for approximately two weeks. The wells are checked for evaporationof medium and brought back to 200 μl per well as necessary during thisprocess. When a large percentage of the colonies in the plate are nearconfluency, 100 μl of medium is collected from each well for analysis bydot blot, and the cells are fed with fresh selection medium. Thesupernatant is applied to a nitrocellulose filter in a dot blotapparatus, and the filter is treated at 100° C. in a vacuum oven todenature the protein. The filter is incubated in 625 mM Tris-glycine, pH9.1, 5mM β-mercaptoethanol, at 65° C., 10 minutes, then in 2.5% non-fatdry milk in Western A Buffer (0.25% gelatin, 50 mM Tris-HCl pH 7.4, 150mM NaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at 4° C. on arotating shaker. The filter is incubated with the antibody-HRP conjugatein 2.5% non-fat dry milk in Western A buffer for 1 hour at roomtemperature on a rotating shaker. The filter is then washed three timesat room temperature in PBS plus 0.01% Tween 20, 15 minutes per wash. Thefilter is developed with chemiluminescence reagents (ECL™ directlabelling kit; Amersham Corp., Arlington Heights, Ill.) according to themanufacturer's directions and exposed to film (Hyperfilm ECL, AmershamCorp.) for approximately 5 minutes. Positive clones are trypsinized fromthe 96-well dish and transferred to 6-well dishes in selection mediumfor scaleup and analysis by Western blot.

Example 6

[0155] Full-length zlmda33 protein is produced in BHK cells transfectedwith pZMP6/zlmda33 (Example 4). BHK 570 cells (ATCC CRL-10314) areplated in 10-cm tissue culture dishes and allowed to grow toapproximately 50 to 70% confluence overnight at 37° C., 5% CO₂, inDMEM/FBS medium (DMEM, Gibco/BRL High Glucose; Life Technologiessupplemented with 5% fetal bovine serum (Hyclone, Logan, Utah), 1 mML-glutamine (JRH Biosciences, Lenexa, Kans.), and 1 mM sodium pyruvate(Life Technologies)). The cells are then transfected with pZMP6/zlmda33by liposome-mediated transfection (using Lipofectamine™; LifeTechnologies), in serum free (SF) medium (DMEM supplemented with 10mg/ml transferrin, 5 mg/ml insulin, 2 mg/mil fetuin, 1% L-glutamine and1% sodium pyruvate). The plasmid is diluted into 15-ml tubes to a totalfinal volume of 640 μl with SF medium. 35 μl of the lipid mixture ismixed with 605 μl of SF medium, and the resulting mixture is allowed toincubate approximately 30 minutes at room temperature. Five millilitersof SF medium is then added to the DNA:lipid mixture. The cells arerinsed once with 5 ml of SF medium, aspirated, and the DNA:lipid mixtureis added. The cells are incubated at 37° C. for five hours, then 6.4 milof DMEM/10% FBS, 1% PSN media is added to each plate. The plates areincubated at 37° C. overnight, and the DNA:lipid mixture is replacedwith fresh 5% FBS/DMEM medium the next day. On day 5 post-transfection,the cells are split into T-162 flasks in selection medium (DMEM+5% FBS,1% L-Gln, 1% sodium pyruvate, 1 μM methotrexate). Approximately 10 dayspost-transfection, two 150-mm culture dishes of methotrexate-resistantcolonies from each transfection are trypsinized, and the cells arepooled and plated into a T-162 flask and transferred to large-scaleculture.

Example 7

[0156] cDNAs and cDNA libraries from a variety of cells and tissues werescreened for zlmda33 sequences by PCR using conventional procedures.Cells and tissues testing positive included fetal brain, spinal cord,testis, prostate smooth muscle cells, thyroid, ovarian tumor, uterinetumor, and bone marrow. A tentative positive signal was found inesophageal tumor. Cells and tissues testing negative included adrenalgland, bladder, brain, cervix, colon, fetal heart, fetal kidney, fetalliver, fetal lung, fetal muscle, fetal skin, heart, kidney, liver, lung,lymph node, mammary gland, melanoma, ovary, pancreas, pituitary,placenta, prostate, rectum, salivary gland, skeletal muscle, smallintestine, spleen, stomach, thymus, trachea, uterus, adipocyte, islet,bone, liver tumor, lung tumor, rectal tumor, stomach tumor, and K562(human chronic myelogenous leukemia), RPMI 1788 (B-cell), W138 (lungfibroblast), CD3+, HaCAT (keratinocyte), HPV (prostate epithelia), HPVS(prostate epithelia), and MG63 (osteosarcoma) cell lines.

[0157] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 6 1 1485 DNA Homo sapiens CDS (174)...(920) 1 ctcattcttc ttgctcaaattttccaagct tggctaccac gcaaatgaat ccacccaaac 60 gtcaccaagt ggagcagggtaccagtatag gtgcaaaaac accctcaatt ccaggagctc 120 cacactgaat tcaggccagtccctggaacc tccccagttt ggctaccaca caa atg 176 Met 1 aat cca ccc aaa cgtcgc caa gtg gag cag ggt ccc agt aca ggt gca 224 Asn Pro Pro Lys Arg ArgGln Val Glu Gln Gly Pro Ser Thr Gly Ala 5 10 15 aaa aaa ccc tca att tcagga gct cca cac ctg aat tca tac cag tcc 272 Lys Lys Pro Ser Ile Ser GlyAla Pro His Leu Asn Ser Tyr Gln Ser 20 25 30 ctg gaa ctt ccc cag aag cagcag gat tct ggc act gag gag ctc atg 320 Leu Glu Leu Pro Gln Lys Gln GlnAsp Ser Gly Thr Glu Glu Leu Met 35 40 45 ata gtc ctg gaa caa ggg aca gaagtg agg ttg agc ctg gaa gag gtc 368 Ile Val Leu Glu Gln Gly Thr Glu ValArg Leu Ser Leu Glu Glu Val 50 55 60 65 atc ctc atc ttg gcc cca gag acagtg ctg cag ctg acc ctg gag aac 416 Ile Leu Ile Leu Ala Pro Glu Thr ValLeu Gln Leu Thr Leu Glu Asn 70 75 80 aca gtc ctt gtg att gtc cct gag catgtc ctg agg tca gaa gat ggc 464 Thr Val Leu Val Ile Val Pro Glu His ValLeu Arg Ser Glu Asp Gly 85 90 95 ctg cag tcc cct gtg cag atc cag tac atcata cct tcc gtt gat gac 512 Leu Gln Ser Pro Val Gln Ile Gln Tyr Ile IlePro Ser Val Asp Asp 100 105 110 ttc agc ttg gag ttc cat gct caa gat ggagac atc tca gac atg aga 560 Phe Ser Leu Glu Phe His Ala Gln Asp Gly AspIle Ser Asp Met Arg 115 120 125 aga gag aat gtg cct ttt tca cct gca gaagaa ggg aag gca gca ccc 608 Arg Glu Asn Val Pro Phe Ser Pro Ala Glu GluGly Lys Ala Ala Pro 130 135 140 145 ctg tat cag cag ccc ttg atg ata ccccaa gca aac cac atg gct ggg 656 Leu Tyr Gln Gln Pro Leu Met Ile Pro GlnAla Asn His Met Ala Gly 150 155 160 atc agc cct tct ttc cta gta acc ccattg tgc att cca cgc tgt cgg 704 Ile Ser Pro Ser Phe Leu Val Thr Pro LeuCys Ile Pro Arg Cys Arg 165 170 175 gca gcc ttc ccc caa tgc tac cct ctacca ccc aca cct agt ccc gtg 752 Ala Ala Phe Pro Gln Cys Tyr Pro Leu ProPro Thr Pro Ser Pro Val 180 185 190 gga cgc cct aga cca gcc gac tcc agtttc agc ctg cat ggt atg gag 800 Gly Arg Pro Arg Pro Ala Asp Ser Ser PheSer Leu His Gly Met Glu 195 200 205 ctc ttg tgc acc tcc tcc ctc aga cctatg ccc cct tca cca agt cct 848 Leu Leu Cys Thr Ser Ser Leu Arg Pro MetPro Pro Ser Pro Ser Pro 210 215 220 225 ggt ccc cag gtc tat cac agg gttcac cat agg cct ccc agc agg gca 896 Gly Pro Gln Val Tyr His Arg Val HisHis Arg Pro Pro Ser Arg Ala 230 235 240 cgg aga tgt ctc ttt agg aag tgatttaacccaa gagccacccc ctgcattgat 950 Arg Arg Cys Leu Phe Arg Lys * 245aggtcagaga ttgtccagat ccttagtcag tgcattctct gaaatgtgga gagaaagtaa 1010tttgaccact tgcttgccct ttgctgttcc ccatcatcaa ccactgtctt caacagcgga 1070gggtcccaga tgctgcaggg agggggagaa ctgcagggag ttcaaataaa acattcacat 1130ttcacttcac acacactgtc ccttagactt tctcttccta tttaagcaca tacatccaac 1190cacactcaat caaatccctg actgctccat gtgagagttc tgcttccagc atgacgtggt 1250ctgaaagttc atctgaagac agctgctcac tcccgggggc taacaccacc ccttgcatgc 1310tgatgtcctt gtagtcattg gtctgatgcc acaataaata attcctaagg ctgatgctct 1370atttctgccc tgagactctc ccctttttct ccaagctgtg ccccattcct tgtcttagtc 1430caggttccct acactcccca ggccaatgct tttgaataaa tcttgacgtc attga 1485 2 248PRT Homo sapiens 2 Met Asn Pro Pro Lys Arg Arg Gln Val Glu Gln Gly ProSer Thr Gly 1 5 10 15 Ala Lys Lys Pro Ser Ile Ser Gly Ala Pro His LeuAsn Ser Tyr Gln 20 25 30 Ser Leu Glu Leu Pro Gln Lys Gln Gln Asp Ser GlyThr Glu Glu Leu 35 40 45 Met Ile Val Leu Glu Gln Gly Thr Glu Val Arg LeuSer Leu Glu Glu 50 55 60 Val Ile Leu Ile Leu Ala Pro Glu Thr Val Leu GlnLeu Thr Leu Glu 65 70 75 80 Asn Thr Val Leu Val Ile Val Pro Glu His ValLeu Arg Ser Glu Asp 85 90 95 Gly Leu Gln Ser Pro Val Gln Ile Gln Tyr IleIle Pro Ser Val Asp 100 105 110 Asp Phe Ser Leu Glu Phe His Ala Gln AspGly Asp Ile Ser Asp Met 115 120 125 Arg Arg Glu Asn Val Pro Phe Ser ProAla Glu Glu Gly Lys Ala Ala 130 135 140 Pro Leu Tyr Gln Gln Pro Leu MetIle Pro Gln Ala Asn His Met Ala 145 150 155 160 Gly Ile Ser Pro Ser PheLeu Val Thr Pro Leu Cys Ile Pro Arg Cys 165 170 175 Arg Ala Ala Phe ProGln Cys Tyr Pro Leu Pro Pro Thr Pro Ser Pro 180 185 190 Val Gly Arg ProArg Pro Ala Asp Ser Ser Phe Ser Leu His Gly Met 195 200 205 Glu Leu LeuCys Thr Ser Ser Leu Arg Pro Met Pro Pro Ser Pro Ser 210 215 220 Pro GlyPro Gln Val Tyr His Arg Val His His Arg Pro Pro Ser Arg 225 230 235 240Ala Arg Arg Cys Leu Phe Arg Lys 245 3 6 PRT Artificial Sequence peptidetag 3 Glu Tyr Met Pro Met Glu 1 5 4 744 DNA Artificial Sequencedegenerate nucleotide sequence 4 atgaayccnc cnaarmgnmg ncargtngarcarggnccnw snacnggngc naaraarccn 60 wsnathwsng gngcnccnca yytnaaywsntaycarwsny tngarytncc ncaraarcar 120 cargaywsng gnacngarga rytnatgathgtnytngarc arggnacnga rgtnmgnytn 180 wsnytngarg argtnathyt nathytngcnccngaracng tnytncaryt nacnytngar 240 aayacngtny tngtnathgt nccngarcaygtnytnmgnw sngargaygg nytncarwsn 300 ccngtncara thcartayat hathccnwsngtngaygayt tywsnytnga rttycaygcn 360 cargayggng ayathwsnga yatgmgnmgngaraaygtnc cnttywsncc ngcngargar 420 ggnaargcng cnccnytnta ycarcarccnytnatgathc cncargcnaa ycayatggcn 480 ggnathwsnc cnwsnttyyt ngtnacnccnytntgyathc cnmgntgymg ngcngcntty 540 ccncartgyt ayccnytncc nccnacnccnwsnccngtng gnmgnccnmg nccngcngay 600 wsnwsnttyw snytncaygg natggarytnytntgyacnw snwsnytnmg nccnatgccn 660 ccnwsnccnw snccnggncc ncargtntaycaymgngtnc aycaymgncc nccnwsnmgn 720 gcnmgnmgnt gyytnttymg naar 744 5248 PRT Homo sapiens 5 Met Asn Pro Pro Lys Arg Arg Gln Val Glu Gln GlyPro Ser Thr Gly 1 5 10 15 Ala Lys Lys Pro Ser Ile Ser Gly Ala Pro HisLeu Asn Ser Tyr Gln 20 25 30 Ser Leu Glu Leu Pro Gln Lys Gln Gln Asp SerGly Thr Glu Glu Leu 35 40 45 Met Ile Val Leu Glu Gln Gly Thr Glu Val ArgLeu Ser Leu Glu Glu 50 55 60 Val Ile Leu Ile Leu Ala Pro Glu Thr Val LeuGln Leu Thr Leu Glu 65 70 75 80 Asn Thr Val Leu Glu Ile Val Pro Glu HisVal Leu Arg Ser Glu Asp 85 90 95 Gly Leu Gln Ser Pro Val Gln Ile Gln TyrIle Ile Pro Ser Ile Asp 100 105 110 Asp Phe Ser Leu Glu Phe His Ala GlnAsp Gly Asp Ile Ser Asp Met 115 120 125 Arg Arg Glu Asn Val Pro Phe SerPro Ala Glu Glu Gly Lys Ala Ala 130 135 140 Pro Leu Tyr Gln Gln Pro LeuMet Ile Pro Gln Ala Asn His Met Ala 145 150 155 160 Gly Ile Ser Pro SerPhe Leu Val Thr Pro Leu Cys Ile Pro Arg Cys 165 170 175 Arg Ala Ala PhePro Gln Cys Tyr Pro Leu Pro Pro Thr Pro Ser Pro 180 185 190 Val Gly ArgPro Arg Pro Ala Asp Ser Ser Phe Ser Leu His Gly Met 195 200 205 Glu LeuLeu Cys Thr Ser Ser Leu Arg Pro Met Pro Pro Ser Pro Ser 210 215 220 ProGly Pro Gln Val Tyr His Arg Val His His Arg Pro Pro Ser Arg 225 230 235240 Ala Arg Arg Cys Leu Phe Arg Lys 245 6 744 DNA Artificial Sequencedegenerate nucleotide sequence 6 atgaayccnc cnaarmgnmg ncargtngarcarggnccnw snacnggngc naaraarccn 60 wsnathwsng gngcnccnca yytnaaywsntaycarwsny tngarytncc ncaraarcar 120 cargaywsng gnacngarga rytnatgathgtnytngarc arggnacnga rgtnmgnytn 180 wsnytngarg argtnathyt nathytngcnccngaracng tnytncaryt nacnytngar 240 aayacngtny tngarathgt nccngarcaygtnytnmgnw sngargaygg nytncarwsn 300 ccngtncara thcartayat hathccnwsnathgaygayt tywsnytnga rttycaygcn 360 cargayggng ayathwsnga yatgmgnmgngaraaygtnc cnttywsncc ngcngargar 420 ggnaargcng cnccnytnta ycarcarccnytnatgathc cncargcnaa ycayatggcn 480 ggnathwsnc cnwsnttyyt ngtnacnccnytntgyathc cnmgntgymg ngcngcntty 540 ccncartgyt ayccnytncc nccnacnccnwsnccngtng gnmgnccnmg nccngcngay 600 wsnwsnttyw snytncaygg natggarytnytntgyacnw snwsnytnmg nccnatgccn 660 ccnwsnccnw snccnggncc ncargtntaycaymgngtnc aycaymgncc nccnwsnmgn 720 gcnmgnmgnt gyytnttymg naar 744

What is claimed is:
 1. An isolated polypeptide comprising at least ninecontiguous amino acid residues of SEQ ID NO: 2 or SEQ ID NO:
 5. 2. Thepolypeptide of claim 1 wherein said at least nine contiguous amino acidresidues comprise residues 2-8, 15-22, 38-45, 94-99, 110-115, 196-202,215-220, or 235-241 of SEQ ID NO:
 2. 3. The polypeptide of claim 1wherein said at least nine contiguous amino acid residues compriseresidues 2-22, 110-133, 120-133, 121-132, 192-203, 214-226, or 214-242of SEQ ID NO: 2
 4. The isolated polypeptide of claim 1 which is from 15to 1500 amino acid residues in length.
 5. The isolated polypeptide ofclaim 1 wherein said at least nine contiguous amino acid residues of SEQID NO: 2 or SEQ ID NO: 5 are operably linked via a peptide bond orpolypeptide linker to a second polypeptide selected from the groupconsisting of maltose binding protein, an immunoglobulin constantregion, a polyhistidine tag, and a peptide as shown in SEQ ID NO:
 3. 6.The isolated polypeptide of claim 1 comprising at least 30 contiguousresidues of SEQ ID NO: 2 or SEQ ID NO:
 5. 7. An expression vectorcomprising the following operably linked elements: a transcriptionpromoter; a DNA segment encoding a protein comprising residues 1-248 ofSEQ ID NO: 2 or SEQ ID NO: 5; and a transcription terminator.
 8. Theexpression vector of claim 7 further comprising a secretory signalsequence operably linked to the DNA segment.
 9. The expression vector ofclaim 7 wherein the DNA segment comprises nucleotides 174-917 of SEQ IDNO:
 1. 10. A cultured cell into which has been introduced the expressionvector of claim 7, wherein the cell expresses the DNA segment.
 11. Amethod of making a protein comprising: culturing the cell of claim 10under conditions whereby the DNA segment is expressed and the protein isproduced; and recovering the protein.
 12. The method of claim 11 whereinthe expression vector further comprises a secretory signal sequenceoperably linked to the DNA segment and wherein the polypeptide issecreted into and recovered from a medium in which the cell is cultured.13. A protein produced by the method of claim
 11. 14. An antibody thatspecifically binds to the protein of claim
 13. 15. The antibody of claim14 which is labeled to produce a detectable signal.
 16. A method ofdetecting, in a test sample, a polypeptide selected from the groupconsisting of: (a) a polypeptide as shown in SEQ ID NO: 2; (b) apolypeptide as shown in SEQ ID NO: 5; and (c) proteolytic fragments of(a) or (b), the method comprising combining the test sample with theantibody of claim 14 under conditions whereby the antibody binds to thepolypeptide, and detecting the presence of antibody bound to thepolypeptide.
 17. An isolated polynucleotide comprising nucleotides 1-744of SEQ ID NO: 4 or SEQ ID NO:
 6. 18. The isolated polynucleotide ofclaim 17 comprising nucleotides 174-917 of SEQ ID NO: 1.